RU2794777C1 - Modified nutrient medium for cultivation of potato plants based on murashige-skoog agar nutrient medium and method for growing potato plants under aseptic conditions using this nutrient medium - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is related to new nanocomposites (NC) based on natural polysaccharides arabinogalactan (I), sulphate arabinogalactan (II) and κ-carrageenan (III) with manganese-containing nanoparticles, as well as to a method of growing plants using NC when grown under aseptic conditions on a modified agar nutrient medium containing NC. The invention can be used as a component of low-dose organo-mineral fertilizers with prolonged action.

EFFECT: inclusion of NC in the composition of the agar nutrient medium makes it possible to cultivate planting material with improved morphometric and/or physiological parameters, free from phytopathogens.

2 cl, 12 dwg, 2 tbl, 8 ex

Description

The invention relates to the field of bio- and nanotechnologies of macromolecular chemistry and microbiology in crop production, specifically to new nanocomposites (NC) based on natural polysaccharides arabinogalactan ( I ), arabinogalactan sulfate ( II ) and κ-carrageenan ( III ) with manganese-containing nanoparticles, as well as to a method for growing plants using NK when grown under aseptic conditions on a modified agar nutrient medium containing NK. The proposed invention can be a component of multi-purpose trophic low-dose organo-mineral fertilizers with a prolonged action, and also used as a nutrient medium that stimulates the development and protection of plants and is intended to obtain high-quality planting material for agricultural and horticultural crops used in aeroponic and hydroponic technologies, when carrying out biotechnological research, as well as for the creation of closed life support systems.

Today, agriculture is facing serious problems associated with the need for optimal plant nutrition during the growing season. Taking into account the growing demand of the world market, great efforts are directed to the development of a new generation of complex mineral fertilizers. Such fertilizers should improve the assimilation of microelements by plants in safe doses, ensure their delayed action, and be resistant to leaching of microelements from various types of soils. In addition, every year the search for more efficient and safer fertilizers becomes an increasingly urgent task, especially in the context of climate change and the relevance of healthy nutrition. The use of nanomaterials as microfertilizers and plant protection products helps to increase plant resistance to adverse weather conditions, reduce morbidity and increase stress resistance, such organomineral nanosystems allow you to get more yield from the same areas, and better absorption of nutrients minimizes their loss to the environment.

Advances in the development of new materials and methods of using nanotechnologies to improve the efficiency of agricultural production are widely covered in scientific publications [J.S. Duhan, et al. "Nanotechnology: The new perspective in precision agriculture" // Biotechnology Reports, 2017, V. 15, 11-23; A. Husen, K.S. Siddiqi "Phytosynthesis of nanoparticles: Concept, controversy and application" // Nanoscale Research Letters, 2014, 9(1), 229; G.S. Nechitailo, O.A. Bogoslovskaya, I.P. Ol'khovskaya, N.N. Glushchenko "Influence of iron, zinc, and copper nanoparticles on some growth indices of pepper plants" // Nanotechnologies in Russia, 2018, V. 13, 161-167; L.R. Khot L.R., et al. "Applications of nanomaterials in agricultural production and crop protection: A review" Crop Protection, 2012, V. 35, 64-70]. Modern composite biomaterials, including metal-containing nanoparticles, acquire increased resistance to external influences, change solubility, acquire higher biological activity, etc. [A.D. Pomogailo, G.I. Dzhardimalieva "Nanostructured materials preparation via condensation ways" // Science+Business Media, Dordrecht, 2014; C.N.R. Rao, A. Müller, A.K. Cheetham "The Chemistry of Nanomaterials" // Wiley-VCH, Weinheim, 2004; G.F. Prozorova, et al. "Green synthesis of water-soluble nontoxic polymeric nanocomposites containing silver nanoparticles" // Int. J. Nanomedicine. 2014, 9, 1883-1889; M.V. Lesnichaya, B.G. Sukhov, et al. "Chiroplasmonic magnetic gold nanocomposites produced by one-step aqueous method using κ-carrageenan" // Carbohydr. Polym. 2017, 175, 18-26]. Biopolymer materials are among the most promising as effective stabilizing matrices of nanosized metal particles [Rozenberg B.A., Tenne R. "Polymer-Assisted fabrication of nanoparticles and nanocomposites" // Progress in Polymer Science. 2008, 33, 40-112; Ochsner A., Shokuhfar A. "New Frontiers of Nanoparticles and Nanocomposite Materials. Novel Principles and Techniques" // Springer-Verlag Berlin Heidelberg, Heidelberg, 2013], capable of forming the latest functional materials, including those for the production of fertilizers based on metal-containing NK. At the same time, nanochemistry faces such tasks as the creation of biocompatible, safe and biologically easily degradable nanosubstances, so the use of natural polysaccharides as matrices is more than relevant [A.I., Nozhkina O.A., Ganenko T.V., Graskova I.A., Sukhov B.G., Artem' ev A.V., Trofimov B.A., Krutovsky K.V. Selenium nanocomposites in natural matrices as potato recovery agent. International Journal of Molecular Sciences. 2021, 22, 4576; Lesnichaya M.V., Sukhov B.G., Aleksandrova G.P., Gasilova E.R., Vakul’skaya T.I., Khutsishvili S.S., Sapozhnikov A.N., Klimenkov I.V., Trofimov B.A. Chiroplasmonic magnetic gold nanocomposites produced by one-step aqueous method using κ-carrageenan. Carbohydrate Polymers. 2017, 175, 18-26; Ganenko T.V., Tantsyrev A.P., Sapozhnikov A.N., Khutsishvili S.S., Vakul’skaya T.I., Fadeeva T.V., Sukhov B.G., Trofimov B.A. Nanocomposites of silver with arabinogalactan sulfate: Preparation, structure, and antimicrobial activity. Russian Journal of General Chemistry. 2015, 85, 477-484].

Nanomaterials used in various methods of healing and cultivation of planting material are a promising direction and are designed to stimulate plant growth when grown in open or protected ground conditions. At the same time, the modern development of biotechnology is inextricably linked with the cultivation of plants on artificial nutrient media, which have a balanced composition of nutrients necessary for the full growth and development of plants in large quantities, especially plant forms that are difficult to propagate under normal conditions. Of great importance is the use of artificial nutrient media in the development of autonomous life support systems, for example, in the conditions of long-term space flights. All this involves the use of high-tech methods, the improvement of plant cultivation techniques based on safe and biodegradable materials in order to obtain planting material free from various phytopathogens. This means that an important characteristic is the production of multi-purpose nanocomposites that inhibit the formation of biofilms and exhibit an antimicrobial effect against phytopathogens, but at the same time are safe for representatives of soil microflora. Thus, the bacterium Clavibacter michiganensis ( Cms ) in most countries of the world is a quarantine object and causes serious crop losses (about half) [Eichenlaub R., Gartemann K.-H. "The Clavibacter michiganensis subspecies: Molecular investigation of gram-positive bacterial plant pathogens" // Annual Review, Phytopathology, 2011, V. 49, 445-464; Li X., Tambong J., Yuan KX, Chen W., Xu H., Lévesque CA, De Boer SH "Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses" // International Journal of Systematic Evolutinary Microbiology, 2018, V. 68, 234-240]. The infection is latent and manifests itself in the form of wilt and yellowing of the stems during the growing season. It can be detected only with a longitudinal section of the tuber in the form of a ring, while outwardly the tuber used as seed material may look absolutely healthy. The problem is exacerbated by the lack of effective methods to combat this bacterium, all measures are only preventive and are associated with the processing of inventory and manual removal of diseased plants from the fields. Therefore, the search for an agent to regulate the abundance of this pathogen is extremely important. No less relevant is the search for environmentally friendly substances used to improve the health of cultivated plants. Therefore, an essential characteristic of new nanocomposites is their impact on the environment, in particular, on the viability of representatives of soil microflora.

An invention is known [RU 2601757 C1, publication date 11/10/2016], which can be used in agriculture as a composition with antimicrobial and antitoxic effects and containing a binary mixture of a colloidal solution of nanostructured silver particles with a particle size of 2-100 nm, a stabilizer and a solvent . To obtain a composition, a non-polar solvent from the group of saturated hydrocarbons or water is used as a solvent. The composition contains an aqueous solution of a hydro-containing polymer as a stabilizer. The composition additionally contains a solution of a water repellent agent based on organosilicon compounds in the form of siloxanes or siliconates in an aqueous dispersion or in a non-polar solvent. EFFECT: invention makes it possible to obtain environmentally safe stable binary colloidal mixtures with high biological activity. The disadvantage of the invention is the use in the proposed method of an expensive component in the form of silver nanoparticles, in addition, the nanoparticles will exhibit a cytotoxic effect in relation to the natural microbiome.

Nanofertilizers are described in the form of metallic nanoparticles [WO 2013121244 A1, publication date 08/22/2013], which are coated with substances that are nutritional microadditives or their precursors. According to the invention, at least one component from a wide range of substances, including compounds of carbon, phosphorus, nitrogen, boron and other elements necessary for plant nutrition and development, as well as salts, chelates and oxides of metals, and other compounds. Just like in the previous analogue, this technical solution uses nanoparticles of expensive noble metals, such as silver, gold, platinum, etc. its independent role as a nutritional agent is difficult to assess.

The invention described in [EP 2499107 A1, publication date 09/19/2012] proposes a method for growing plants using nanoparticles, the essence of which is the use of multilayer carbon nanotubes at an effective concentration of 10-200 pg/ml to increase the germination of tomato seeds and increase green plant masses. Tomato plants grown on a nutrient medium in the presence of carbon nanotubes have a larger biomass volume in comparison with the control, while differing from the control in a more developed root system. The authors attribute the improvement in indicators to an increase in the intensity of water absorption processes in seeds in the presence of carbon nanotubes. However, it is known that carbon nanotubes, like asbestos, are able to penetrate the human body through the skin and nasopharynx and destroy cells and have a carcinogenic effect. Therefore, the introduction of carbon tubes in agriculture and horticulture requires thorough toxicological studies on their accumulation and removal from plants, as well as additional study of the impact of such nanosubstances on the human body, which makes their use in crop production unlikely in the near future.

The closest analogue is the invention using NCs with stabilized nanoparticles [US 20110123589 A1, publication date 05/26/2011]. This invention describes nanocomposite materials in the form of a three-dimensional structure formed by a polymer matrix consisting of a polysaccharide composition of neutral or anionic polysaccharides and branched cationic polysaccharides, in which metal nanoparticles are uniformly dispersed and stabilized. Obtaining nanosized composite materials based on hydrated polysaccharide compositions in the form of hydrogels or in non-hydrated form formed by mono- or oligosaccharide branched derivatives of chitosan with lactose. Nanoparticles of silver, gold, platinum, palladium, copper, zinc, nickel and their mixtures were chosen as the filler. It was shown that the resulting compositions have a wide range of strong bactericidal activity associated with the nanosize of metal particles and the presence of biological activity of the original matrix, but do not exhibit cytotoxicity. Gels based on the obtained nanocomposites were tested both on gram-negative strains (Escherichia coli, Pseudomonas aeruginosa) and on gram-positive strains (Staphylococcus aureus, Staphylococcus epidermidis). Cytotoxicity tests on various eukaryotic cell lines (osteoblasts (MG63), hepatocytes (HEPG2) and fibroblasts (3T3)) show that these three-dimensional nanocomposite gels are not cytotoxic, even though they retain an effective bactericidal effect. The proposed compositions can be used in the development of a new generation of biomaterials with antimicrobial properties, and for many other applications in biomedical, pharmaceutical and food products. However, as in the analogues described above, the proposed invention uses expensive metals - silver, gold, platinum, etc., and chitosan used as a matrix is poorly soluble in water. In addition, the use of such bactericidal nanomaterials in the food industry in relation to bacterial phytopathogens can also harm the natural microbiome. The indicated size of nanoparticles in the compositions used is in the range from 5 to 150 nm with a controlled average size in the range from 30 to 50 nm, which, in the absence of a narrow dispersion, does not allow obtaining a composite as a truly effective antibacterial agent, since it is known that the smallest nanoparticles , about 10 nm, show the highest antibacterial activity [Raza M.A., Kanwal Z., Rauf A., Sabri A.N., Riaz S., Naseem S. "Size- and shape-dependent antibacterial studies of silver nanoparticles synthesized by wet chemical routes" / / Nanomaterials (Basel, Switzerland), 2016, V. 6 (4), 74].

Based on the combination of features, the invention [WO 2017101691 A1, publication date 06/22/2017] was adopted as a prototype, which describes a method for growing plants using nanoparticles by germinating seeds and then growing plants under aseptic conditions on an agar nutrient medium containing nanoparticles. To implement the method, Murashige-Skoog agar nutrient medium is used, which contains iron, zinc or copper nanoparticles as nanoparticles, or a combination of iron, zinc and copper nanoparticles. However, the nanoparticles used in the invention do not have a coating that would prevent the aggregation of nanoparticles into larger agglomerates, slow down the leaching of trace elements from the soil, thereby not creating an additional environmental burden.

The objective of the present invention is to create multifunctional nanotechnological materials suitable for use as microfertilizers and plant protection products, providing improved biometric and / or physiological parameters of plants, contributing to better absorption of micronutrient fertilizers by plants in safe doses, which would have a delayed effect (resistance to leaching of trace elements for different types of soils), stimulating stress resistance of plants and exhibiting a pronounced antibacterial effect against phytopathogenic bacteria. Development of a method for using such NCs in order to obtain a healthy high-quality planting material.

The problem is solved by using compounds that are manganese-containing NCs based on polysaccharides, and by the method of growing plants under aseptic conditions on a modernized nutrient medium, in which NCs with manganese-containing nanoparticles are used as a nutrient agent.

To implement the present invention, organomineral complexes are proposed, which are water-soluble compounds based on the natural polysaccharide arabinogalactan, sulfated arabinogalactan or κ-carrageenan, which stabilize manganese-containing nanoparticles Mn(OH) ₂ × nH ₂ O with an average distribution of 3-9 nm and represent a multifunctional agent with antimicrobial activity against the phytopathogen Cms , but safe for representatives of soil microflora, stimulate the development and stress resistance of Solanum tuberosum L. potato plants, as well as a method of growing plants using NC under aseptic conditions on a modified agar nutrient medium. In this case, manganese is included in the nutrient medium in the form of NC, consisting of nanoparticles in a composite material (matrix), in turn, the matrices used are non-toxic, biodegradable and water-soluble natural polysaccharides. As the initial basis of the nutrient medium for the implementation of the proposed method, a widely used nutrient medium according to the Murashige-Skoog prescription was chosen, which contains all the necessary components for plant development, namely vitamins, carbohydrates and amino acids, protein hydrolysates, inorganic salts, as well as macro - and trace elements.

The advantages of the proposed invention is the use of simple and environmentally friendly compounds: manganese-containing nanoparticles stimulate the stress resistance of plants due to the antioxidant effect, inhibit the formation of a biofilm and exhibit antimicrobial activity; available natural polysaccharides (the content of arabinogalactan in larch Larixsibirica Ledeb. up to 15%, κ-carrageenan is found in widespread red seaweed), are non-toxic and highly soluble in water. The polysaccharide matrix acts as a gel-forming binding agent, prevents the trace element from being washed out of the soil, and gradually releases manganese for absorption by plants. The inclusion of NC in the composition of the agar nutrient medium makes it possible to cultivate planting material free from phytopathogens. EFFECT: invention provides improved seed germination, as well as obtaining planting material with improved morphometric and/or physiological parameters. The method can be implemented in autonomous life support systems, for example, in conditions of long-term space flights.

For the synthesis of nanocomposites, arabinogalactan obtained from Siberian larch Larixsibirica Ledeb was used. Wood Chemistry Ltd. (Irkutsk, Russia). In turn, sulfated arabinogalactan was obtained by functionalization of the original arabinogalactan according to the method described in the patent [RU 2319707 C1, publication date 03/20/2008]. The polysaccharide κ-carrageenan is derived from red seaweed by CP Celco Comp. (Lille Skensved, Denmark). NCs can be synthesized according to the following procedure: aqueous solutions of manganese sulfate from 0.30 to 0.69 g (in 2 or 3 ml of water) are added to an aqueous solution of polysaccharide with vigorous stirring on a magnetic stirrer, 0.1-0.3 ml of NH ₄ OH (and 0.2 ml of hydrazine in the case of arabinogalactan) and left at room temperature for 3-24 hours. The solution is filtered and the target products are isolated by precipitating the filtrate in ethanol, the precipitate is filtered off and dried in vacuo. The content of manganese in the obtained samples, determined by elemental analysis and atomic absorption analysis, varies within 4.8–20.3% depending on the reaction conditions and the matrix used. According to transmission electron microscopy, the size of the nanoparticles varies in the range of 2-20 nm with an average distribution of 3-9 nm (Fig. 1).

The results of the conducted studies demonstrate the absence of a negative effect of NC on the development of plants; biometric indicators are comparable or superior to control samples. In plants grown on media with the introduction of nanocomposites I or II , the root system is extensively developed, yellow leaves are noted only in 10% of plants. The most viable plants are in the variant with III . All NCs ( I - III ) stimulate the stress resistance of potato plants. III has a pronounced antiradical effect from a number of substances under study. Treatment of infected plants with III reduces the content of diene conjugates (DC) and malondialdehyde (MDA) in tissues, indicating the intensity of destructive processes under the influence of reactive oxygen species (ROS). The antibacterial effect against the phytopathogenic bacterium Cms is manifested in all NCs ( I - III ). A pronounced negative effect on the growth of bacteria is revealed when II is introduced with a manganese concentration of 0.00625%. The maximum number of dead cells was noted in samples II and III at concentrations of 0.00625%. The conducted studies have shown that NAs change the morphology of Cms bacterial cells. After incubation of bacteria with I - III , their cell wall is destroyed, the contents of the cell are released to the outside, which leads to the death of the pathogenic bacterium. All NCs I - III show no bactericidal effect on the growth of rhizospheric bacterial cultures A. guillouiae , R. erythropolis , and P. oryzihabitans , and thus NCs I - III are safe for representatives of the natural soil microbiome.

The invention is illustrated by the following examples.

Physicochemical studies were carried out at the Chemical Research Center for Shared Use of the Siberian Branch of the Russian Academy of Sciences (N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences) and the Baikal Analytical Center for Shared Use (Irkutsk Institute of Chemistry named after A.E. Favorsky, Siberian Branch of the Russian Academy of Sciences). Biological studies were carried out using the collections of the Bioresource Center for Collective Use (Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch, Russian Academy of Sciences).

Example #1. Synthesis of a nanocomposite based on arabinogalactan (I).

2.0 g of arabinogalactan (18 kDa) was dissolved in 5 ml of _H2O , 0.40 g of manganese sulfate _MnSO4 × _5H2O in 3 ml of _H2O was added to the resulting solution, and 0.1 ml of NH4OH and 0.2 ml of hydrazine were added with stirring on a magnetic stirrer. After 3 hours of stirring, the reaction mixture was precipitated in alcohol. The product was isolated and purified by precipitating the filtrate in ethanol, followed by separation of the precipitate and drying. The yield of product I was 1.68 g; its manganese content was 5.2%. EPR spectrum: g = 2.090, ΔH = 550 G, A / B = 0.7, N = ¹⁰²⁰ spin/g. IR spectrum (ν, cm ^-1 ): 3425, 2924, 2151, 2071, 1713, 1634, 1423, 1367, 1225, 1152, 1079, 1044, 885, 776, 711, 606, 570, 534, 424.

Example #2. Synthesis of a nanocomposite based on sulfated arabinogalactan (II).

2.0 g of sulfated arabinogalactan (22 kDa) was dissolved in 6 ml of _H2O , 0.30 g of manganese sulfate _MnSO4 × _5H2O in 2 ml of H2O was added to the resulting _solution , and 0.2 ml of _NH4OH was added with stirring on a magnetic stirrer. After 4 hours of stirring, the reaction mixture was precipitated in alcohol. The product was isolated and purified by precipitating the filtrate in ethanol, followed by separation of the precipitate and drying. The yield of product II was 1.80 g; its manganese content was 4.8%. EPR spectrum: g = 2.060, ΔH = 220 G, A / B = 0.8, N = ¹⁰²⁰ spin/g. IR spectrum (ν, cm ^-1 ): 3427, 3264, 3072, 2927, 2897, 2484, 2052, 1705, 1633, 1470, 1433, 1375, 1357, 1214, 1134, 1081, 894, 772, 7 11,616 , 579, 427.

Example #3. Synthesis of a nanocomposite based on κ-carrageenan (III).

3.0 g of κ-carrageenan (1100 kDa) was kept under stirring in 150 ml of H ₂ O while heating at 50°C to a homogeneous medium, 0.69 g of manganese sulfate MnSO ₄ × 5H ₂ O in 3 ml of H ₂ O and 0.3 ml of _NH4OH . After 24 h, the reaction mixture was precipitated in alcohol. The product was isolated and purified by precipitating the filtrate in ethanol, followed by separation of the precipitate and drying. The yield of product III was 2.40 g; its manganese content was 20.3%. EPR spectrum: g = 2.060, ΔH = 240 G, A / B = 1.0, N = ¹⁰²⁰ spin/g. IR spectrum (ν, cm ^-1 ): 3438, 2958, 2921, 2084, 1698, 1635, 1423, 1375, 1261, 1233, 1194, 1149, 1119, 1082, 975, 931, 845, 770, 733 , 702 , 611, 553, 446.

Fig. Fig. 1. Transmission electron microscopy of (A) I , (B) II , and (C) III nanocomposites and histograms with the size distribution of manganese-containing nanoparticles.

Example number 4. Modification of the Murashige-Skoog culture medium using NK I-III

The nutrient medium is prepared on the basis of a widely used standardized agar nutrient medium according to the Murashige-Skoog recipe, which contains all the necessary components for plant development, namely vitamins, carbohydrates and amino acids, protein hydrolysates, inorganic salts, as well as macro- and microelements. All components are prepared and added in accordance with the required concentrations, according to the procedure for preparing a nutrient medium according to the prescription [Murashing T., Skoog F. "A received medium for rapid growth and bio-assays with tobacco tissue culture" // Physiologia Plantarum, 1962, V. 15, 473-497]. In this case, manganese is introduced into the nutrient medium not in ionic form (in the form of a salt solution of crystalline manganese sulfate MnSO ₄ × 5H ₂ O (24.1 g/l)), but in the form of NC ( I, II or III ) based on natural arabinogalactan polysaccharides, sulfated arabinogalactan or κ -carrageenan with manganese-containing nanoparticles Mn(OH) ₂ ×nH ₂ O with an average distribution of 3-9 nm. The synthesized NC is dissolved in water with stirring with a magnetic stirrer until complete dissolution, filtered and then added to the nutrient medium. The amount of NC is taken from the calculation of the required content of manganese in the medium in accordance with the prescription (in terms of the mass amount of elemental manganese required for introduction into the medium - 5.49 g/l) and based on the mass fraction of metal in the NC.

Example number 5. Study of stimulation of plant development in the presence of nanocomposites.

To study the development of plants in the presence of nanocomposites, potato plants of the Lukyanovsky variety, susceptible to biotic stresses, were used in vitro [Romanenko AS, Riffel AA, Graskova IA, Rachenko MA "The role of extracellular pH-homeostasis in potato resistance to ring rot pathogen" / / Journal of Phytopathology, 1999, V. 147, 679-686]. To study the effect on the vegetation and productivity of plants, potato plants were cultivated under factorostatic conditions on a Murashige-Skoog nutrient medium modified with manganese-containing NCs based on natural polysaccharides.

For control samples, manganese was added to the agar nutrient medium in the form of manganese sulfate hydrate MnSO ₄ × 5H ₂ O in the amount according to the Murashige-Skoog nutrient medium recipe. The results were subjected to statistical analysis using a package of statistical add-ons MS Excel, comparison with the control was evaluated by the Mann-Whitney test.

The cuttings were planted at the depth of the internode in an agar nutrient medium and cultivated for 28 days under factorostatic conditions at a temperature of 24–25°C, illumination of 5–6 kLx, and a photoperiod of 16 h, periodically measuring the length and counting the number of leaves. At the end of the experiment, the biomass of the aerial part and the biomass of the roots were determined, and physicochemical analyzes of the plant material were carried out. Independent experiments were performed in triplicate, ten plants were grown in each variant. Three plants from each variant were used for physicochemical experiments.

The results of the experiment showed the absence of a negative effect of NC on the development of potatoes, biometric indicators are comparable or superior to control samples (Fig. 2 and 3). On the 28th day of incubation, the control root system was often well developed, single plants with callus were noted, withered and yellowish leaves appeared. In plants grown on media with the introduction of nanocomposites I or II , the root system was developed extensively, yellow leaves were noted only in 10% of plants. The most viable plants were in the variant with III . They had a well-developed root system, the number of wilted and yellow leaves in potatoes of this variant was less in comparison with the control, callus was not noted. EPR studies have shown that in all plant tissues (roots, stems, leaves) grown in a medium with manganese-containing nanocomposites, as well as in the control, a characteristic multiplet from manganese ions Mn ²⁺ is observed. The spectral characteristics of the signals ( g factor in the region of 2.0031(2), constant (A) 94(2) G) are almost identical to the signals observed in the spectra of control plant biomaterials. Comparison with control shows similar values for the content of manganese in plant organs (Table 1). At the same time, NCs stimulated the development of plants; nanocomposites II and III had the maximum effect. Their introduction into the potato cultivation medium stimulated the development of a powerful root system.

Fig. Fig. 2. Dynamics of biometric indicators of plant length (A) and number of leaves (B) in control and plants grown on a nutrient medium with nanocomposites; 1 - control, 2 - without manganese sulfate in the medium, 3 - I , 4 - II , 5 - III ; * p ≤ 0.05 and ** p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Fig. Fig. 3. Weight of the vegetative part of plants (A) and biomass of roots (B) in control and plants grown on a nutrient medium with nanocomposites; 1 - control, 2 - without manganese sulfate in the medium, 3 - I , 4 - II , 5 - III ; * p ≤ 0.05 and ** p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Table 1. Accumulation of manganese (wt.% ±SD) in plants grown on the medium with nanocomposites ( I-III ) compared with the control. Roots stems Leaves Control 0.17 ± 0.03 0.19 ± 0.04 0.20 ± 0.13 I 0.28 ± 0.10* 0.27 ± 0.01* 0.24 ± 0.20 II 0.05 ± 0.04* 0.16 ± 0.11 0.17 ± 0.13 III 0.29 ± 0.11* 0.38 ± 0.13* 0.24 ± 0.11

* p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Example number 6. Study of stress resistance of plants.

To assess the stress state of plants under the influence of nanocomposites I - III , the following biochemical parameters were studied: peroxidase activity in potato leaf tissues, ROS content, and the amount of lipid peroxidation products (LPO) - DC and MDA in root and leaf tissues. To identify areas of ROS production, plants were infected with Cms , kept for 4 days, then treated with a nanocomposite, and samples for analysis were prepared after 1 h of co-incubation. Plant root tissue samples were incubated for 30 minutes with 5 μM CellROX Deep Red reagent (abs/emm 644/665 nm) (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) dissolved in phosphate buffer. The tissue was then fixed with 2% paraformaldehyde for 15 min. The resulting preparations were embedded in ProLong Gold anti-fader reagent (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and examined using a laser confocal scanning microscope (LKSM) CLSM 710 (Carl Zeiss, Jena, Germany). The study used lasers 405, 561 nm and filters Ch1 410-522. The content of ROS in potato roots was determined spectrophotometrically using xylenol orange dye [Bindschedler LV, Minibayeva F., Gardner SL, Gerrish C., Davies DR, Bolwell GP "Early signaling events in apoplastic oxidative burst in suspension cultured french bean cells involve camp and Ca ²⁺ " // New Phytologist, 2001, 151, 185-194]. Determination of the primary products of LPO and DC in the tissues of potato plants was carried out according to the standard method using hexane and isopropanol 30 and 60 minutes after the addition of the nanocomposite solution to the potato nutrient medium in vitro [Revin VV, Gromova NV, Revina ES, Samonova A.Yu., Tychkov A.Yu., Bochkareva SS, Moskovkin AA, Kuzmenko TP "The influence of oxidative stress and natural antioxidants on morphometric parameters of red blood cells, the hemoglobin oxygen binding capacity, and the activity of antioxidant enzyme" // BioMed Research International, 2019, 2019, 2109269]. The concentration of MDA was determined by the method using 20% trichloroacetic acid and 0.5% solution of thiobarbituric acid [Heath RL, Packer L. "Photoperoxidation in isolated chloroplasts. Kinetics and stoichiometry of fatty acid peroxidation" // Archives of Biochemistry and Biophysics, 1968, 125, 189-198]. Peroxidase activity in potato tissues was determined by the Boyarkin method [Sharlaeva EA, Chirkova V.Yu. "The impact of short-wave UV radiation on peroxidase activity in soft wheat seeds" IOP Conference Series: Earth and Environmental Science, 2021, 670, 012008]. The results were subjected to statistical analysis using a package of statistical add-ons MS Excel, comparison with the control was evaluated by the Mann-Whitney test.

The activity of guaiacol-dependent peroxidase was determined by the Boyarkin method at the end of the period of co-incubation of plants with nanocomposites (Fig. 4). A significant increase in peroxidase activity was found in comparison with the control when growing plants on a medium containing III . The effect of nanocomposites I and II on the activity of the enzyme was not revealed.

To assess the presence of a stress state in plants under the influence of infection, after treatment with nanocomposites and infection in combination with nanocomposites, at the first stage of biochemical studies, we studied the content of ROS in the root tissues of potato plants (Fig. 5), which trigger a cascade of defense programs in the cell, and also capable of inducing lipid peroxidation (LPO).

The products of the damaging effect of ROS on living plant cells at the first stage are DC. Therefore, a series of studies were carried out to assess the amount of DC in the tissues of the roots and leaves of potatoes under the studied stress factors (Fig. 6). It was found that infection of potato with Cms leads to an increase in the content of DC, intermediate products of LPO, both in root tissues and in leaves compared to the control. In case III , the number of DCs in the root tissues increased, while this parameter in the leaves remained at the control level. The addition of III to already infected plants reduced the content of DC both in root tissues and in potato leaves, which indicates a decrease in the intensity of LPO processes. Treatment of healthy plants with nanocomposite II did not affect the DC level. The introduction of this composite to infected plants reduced the amount of DC in root and leaf tissues compared to infected plants without nanocomposite treatment. This effect was most pronounced in the study of samples obtained from plant roots. It was found that I increased the content of DC in the tissues of potato roots by 2 times compared with the control, while the reverse effect was observed in the tissues of the leaves, and a decrease in DC was noted. The application of Composite I to infected plants resulted in a significant increase in the amount of DC in root tissues and a decrease in the amount in leaves compared to infected plants without NA treatment. Thus, all studied NAs reduced the amount of DCs in the leaf tissues of infected plants; II and III also reduced the amount of DCs in the root tissues. The result obtained indicates a decrease in the stress load on the plant under the influence of II and III .

If the stress load on the plant organism does not stop, or even increases, the end product of MDA peroxidation begins to form in the cells, therefore, at the next stage, its content in potato tissues was studied (Fig. 7). It was found that when plants were infected with the Cms pathogen, the MDA content increased both in the roots and in the leaves. Treatment of infection-free plants with Composite III significantly reduced the amount of MDA in both root and leaf tissues. Treatment of infected plants with III led to a decrease in the level of MDA in the studied tissues to the control value. Composite II had no effect on the amount of MDA in both roots and leaves. However, in infected plants significantly increased the amount of MDA in the leaves. Composite I significantly reduced the amount of MDA in the root tissues of both healthy and infected potato plants. In leaf tissues , I increased the content of MDA upon infection with Cms . The result obtained shows that III has a pronounced antiradical effect from a number of the studied substances, treatment of infected plants with it reduced the content of DC and MDA in their tissues, indicating the intensity of destructive processes under the influence of ROS.

Fig. 4. Effect of nanocomposites on the peroxidase activity of potato plants compared with the control group in vitro ; 1 - control, 2 - without manganese sulfate in the medium, 3 - I , 4 - II , 5 - III . Treatment with the nanocomposite did not differ from the control according to the Mann-Whitney test.

Fig. Fig. 5. LKSM micrographs of the effect of nanocomposites on ROS production and corresponding data on the amount of ROS in root tissues in control (A), infected with Cms (B), treated with nanocomposite I (C), treated with nanocomposite I , and infected with Cms (D); * p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Fig. Fig. 6. Influence of nanocomposites on the content of DC in leaves (A) and roots (B) of potato compared with the control group in vitro , as well as in plants infected with the Cms pathogen; 1 - control, 2 - plants infected with Cms , 3 - I without Cms , 4 - infected plants in the presence of I , 5 - II without Cms , 6 - infected plants in the presence of II , 7 - III without Cms , 8 - infected plants in the presence of III ; * p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Fig. Fig. 7. Effect of nanocomposites on the content of MDA in leaves (A) and roots (B) of potatoes compared with the control group in vitro , as well as in plants infected with the Cms pathogen; 1 - control, 2 - plants infected with Cms without nanocomposites, 3 - I without Cms , 4 - infected plants in the presence of I , 5 - II without Cms , 6 - infected plants in the presence of II , 7 - III without Cms , 8 - infected plants in the presence of III ; * p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Example number 7. Determination of the antibacterial effect of manganese-containing nanocomposites I-III.

To do this, a series of experiments was carried out to study the effect of I-III on the viability of bacteria - the causative agent of potato ring rot Cms . Gram-positive bacterium Cms was used, which causes potato ring rot disease (strain Ac-14 05, obtained from the All-Russian Collection of Microorganisms). Bacteria were cultured on GPY medium [Roozen NJM, Van Vuurde JWL "Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry" // Netherlands Journal of Plant Pathology, 1991 , V. 97, 321-334] containing yeast extract 5 g/l (Sigma, USA), glucose 5 g/l (Diam, Russia), peptone 10 g/l (Sigma, USA) , NaCl 5 g/L (NevaReaktiv, Russia) and agar 7 g/L (Diam, Russia). To maintain the culture, tubes with slanted agar medium were placed in a thermostat (at a temperature of 25°C). For the experiment, a bacterial colony was transferred from a solid nutrient medium to a liquid one and grown for 2 days.

A 0.05% aqueous solution of nanocomposites and their precursors was added to flasks with a bacterial suspension (optical density D = 0.9). The solutions of the studied agents were preliminarily subjected to cold sterilization (Minisart NML syringe nozzle, pore size 0.22 μm). The concentration of the active substance was equal for all agents and amounted to 0.000625% and 0.00625% (based on the mass amount of manganese of the precursor substance). When choosing effective concentrations, we were guided by the concentrations used for selenium-containing nanocomposites based on polysaccharides [Perfileva A.I., Nozhkina O.A., Graskova I.A., Sidorov A.V., Lesnichaya M.V., Aleksandrova G.P., Dolmaa G., Klimenkov I.V., Sukhov B.G. "Synthesis of selenium and silver nanobiocomposites and their influence on phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus" // Russian Chemical Bulletin, 2018, V. 67, 157-163], the concentration increased in a 10-fold dose of 0.00625% was used to study cases of local concentration of the substance in the nutrient substrate. To study the bacteriostatic activity of nanocomposites against bacteria, a liquid culture of microorganisms was grown in the dark at 26°C under aerated conditions (80 rpm) in flasks containing a nutrient medium. After the introduction of the composites, the optical density of the suspension was measured at 595 nm using a Bio-Rad spectrophotometer model 680 plate spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) immediately and after 2, 4, 24, 28, 48, 48, 52 and 72 hours of coincubation. The detection of the bactericidal effect of nanocomposites was carried out using the circle method (agar diffusion method) [Sagdic O., Aksoy A., Ozkan G. "Evaluation of the antibacterial and antioxidant potentials of gilaburu (Viburnum opulus L.) fruit extract" // Acta Alimentaria , 2006, v. 35, 487-492].

The effect of nanocomposites on bacterial biofilm formation was studied using the plate method. Microscopy was performed using staining of dead cells with propidium iodide at a final concentration of 7.5 μM (Biotium, USA) [Yamori W., Kogami H., Masuzawa T. "Freezing tolerance in alpine plants as assessed by the FDA-staining method" / / Polar Bioscience, 2005, V. 18, 73-81], during the experiment, bacterial cells are incubated with nanocomposites for 24 hours. The study of bacteria was carried out on an inverted fluorescent microscope AxioObserver Z1 (Carl Zeiss, Germany). Micrographs were taken with an AxioCam MRm3 camera and processed with AxioVision Rel.4.8.2 software.

After incubation with nanocomposites for 1 day, Cms bacteria were fixed in a 2.5% solution of glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h, washed with the same buffer, and additionally fixed with a 2% solution of osmium tetroxide (1–3 h). and embedded in epoxy resin. Sections were made on an Ultracut R ultramicrotome (Leica), which, after contrasting in lead citrate, were examined using a Leo 906 E transmission electron microscope.

The results showed (Fig. 8) that at the initial stage of observation and up to 28 hours, all the applied nanocomposites reduced the growth of bacteria. However, a bacteriostatic effect was further noted when composites with a concentration of 0.00625% manganese in volume were introduced. The most pronounced negative effect on bacterial growth was revealed when II was introduced with a manganese concentration of 0.00625%. The study of the presence of a bactericidal effect in relation to Cms in composites using the method of circles (method of wells, method of diffusion in agar) showed that II had a bactericidal effect in relation to Cms . The zone of bacterial growth inhibition around the well reached 9.0±0.3 mm. In all other variants of the experiment for I and III, no pronounced inhibition of bacterial growth was detected.

For ring rot, biofilm formation is also characteristic, these bacteria are able to accumulate in the conducting channels of the plant, causing them to clog, resulting in wilt of the stems [Secor GA, DeBuhr L., Gudmestad NC Susceptibility of Corynebacterium sepedonicum to disinfectants in vitro // Plant Dis. 1988, 72, 585-588]. The results showed that nanocomposites I and II with a concentration of 0.00625% by volume reduce the biofilm formation of bacteria, and the number of dead cells reaches 30% (Fig. 8). The results showed that in the control sample, the number of dead cells was insignificant (Fig. 9A), when bacteria were treated with nanocomposites at a concentration of 0.000625%, it already led to the appearance of dead cells (Fig. 9 B, D and F). Increasing the concentration of the composites by 10 times resulted in a significant increase in dead bacterial cells (Fig. 9 C, E and G). The maximum number of dead cells was noted in samples II and III at concentrations of 0.00625%. Thus, the results showed the presence of an antibacterial effect in all NCs in relation to the phytopathogenic bacterium Cms . II with a manganese concentration of 0.00625% had the greatest effect. In addition, nanocomposites have been found to change the morphology of Cms bacterial cells. After incubation, the cells thickened and shortened compared to the control (Table 2).

Using transmission electron microscopy, it was shown that Cms bacteria in the control variant, both in transverse (Fig. 10) and longitudinal (Fig. 11) optical sections, had a thick, integral, without pronounced damage to the cell wall. After incubation of bacteria with nanocomposites for 1 h, they lost their native shape and the cell surface became uneven. It was found that after the incubation of bacteria with nanocomposites, their cell wall was destroyed, the contents of the cell were released to the outside, which led to the death of the pathogenic bacterium. Pronounced lysis of the cell wall was noted during the treatment of bacteria with nanocomposites - in the field of view of the microscope in variants with I and II , instead of bacteria, only fragments of their cell wall were often observed (Fig. 11). Treatment of Cms with nanocomposite III resulted in invaginations of the cell wall into the bacterial cell (FIG. 11 G, H), with no adherence of several bacterial cells observed. The obtained micrographs indicate the loss of turgor by bacterial cells and the release of the contents of the cells to the outside.

Fig. Fig. 8. Effect of treatment with nanocomposites with Mn concentrations of 0.000625% and 0.00625% ^† by volume on the growth (A) and biofilm formation (B) of the pathogen Cms ; 1 - control, 2 - I , 3 - I ^† , 4 - II , 5 - II ^† , 6 - III , 7 - III ^† ; * p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Fig. Fig. 9. Micrographs of Cms biofilms for control (A) and in the presence of nanocomposites with Mn concentrations of 0.000625% and 0.00625% ^† by volume for I (B), I ^† (B), II (D), II ^† (E), III ( F), III ^† (G).

Fig. 10. TEM micrographs of Cms bacteria in control (A) and treated with I (B), II (C) and III (D). Transverse optical section of cells.

Fig. 11. TEM micrographs of Cms bacteria in control (A, B) and treated with I (C, D), II (E, F), and III (G, H). Longitudinal optical section of cells.

Table 2. Effect of nanocomposites on the morphological parameters of the Cms bacterium (length, width, and number of dead cells) with manganese concentrations of 0.000625% and 0.00625%† by volume. Length, µm Width, µm Number of dead cells, % Control 3.13 ± 0.08 0.56 ± 0.02 0.34 ± 0.01 I 2.12 ± 0.07* 0.49 ± 0.01* 2.35 ± 0.46* I ^† 2.07 ± 0.08* 0.65 ± 0.19 5.55 ± 0.40* II 2.31 ± 0.09* 0.53 ± 0.01 3.69 ± 0.54* II ^† 2.53 ± 0.34* 0.77 ± 0.01* 24.77 ± 1.70* III 2.41 ± 0.63* 0.62 ± 0.04* 3.07 ± 0.66 III ^† 2.62 ± 0.17* 0.71 ± 0.24* 13.68 ± 4.82* * p ≤ 0.01 compared with the control according to the Mann-Whitney test.

Example number 8. Bactericidal effect of I-III nanocomposites on soil bacteria of the soil microbiome.

Bacteria isolated from the rhizosphere of plants growing in the oil-contaminated territory of the village of Tyret (Zalarinsky district, Irkutsk region, Russia) were used as soil microorganisms. After the isolation of bacteria, their morphological, cultural, physiological and biochemical properties were studied, and the 16S rRNA gene was also sequenced to determine the species [Tretyakova M.S., Belovezhets M.A., Markova Yu.A., Makarova L.E. "The ability of oil-destructing bacteria to reduce the toxic effect of oil on a plant" // Agrochemistry, 2017, No. 12, 56-61].

The Acinetobacter guillouiae strain was isolated from the rhizosphere of the couch grass Elytrigia repens . Cells are short rods, Gram stain negative. Bacteria (aerobes, oxidase- and catalase-positive) form colonies up to 1.2 mm in size, beige, rounded, shiny, slightly convex, homogeneous, soft consistency, with a smooth edge.

The Rhodococcus erythropolis strain was also isolated from the rhizosphere of the grass E. re-pensquack . At an early stage of development, the culture is a rudimentary mycelium, which later breaks down into fragments. The fragments then turn into rods and then into cocci. The bacteria are gram positive. Their colonies are medium, about 1.5-2.5 mm in size. They are cream-colored, dull, pasty, convex, with a rhizoidal margin, rough structure. They are aerobic, oxidase-negative, and have catalase activity.

The Pseudomonas oryzihabitans strain was isolated from the rhizosphere of the sedge Carex hancockiana Maxim. Cells are single or paired straight rods, Gram stain negative. Bacteria (aerobes, catalase- and oxidase-positive, capable of pigmentation) when cultivated on solid media form yellow, smooth, shiny, round with smooth edges, convex, homogeneous, soft consistency, mucous colonies [Silva F. "Pseudomonas (Flavimonas) oryzihabitans" // Revista Chilena de Infectologia, 2015, 32, 445-446].

To clarify the species composition of pure cultures, the nucleotide sequence of the 16S rRNA gene was analyzed, which showed the following nucleotide sequences in size: 1136 bp. (98%), 1429 b.p. (99%) and 1098 b.p. (98%), which identified the generic affiliation to Rhodococcus spp., Pseudomonas spp. and Acinetobacter spp. respectively.

Bacteria were cultivated in the dark for 24 hours on a solid medium consisting of agar, enzymatic hydrolyzate of beef meat, and on a liquid nutrient medium of the same composition. Identification of the possible bactericidal action of nanocomposites was carried out using the circle method (agar diffusion method) [Sagdic O., Aksoy A., Ozkan G. "Evaluation of the antibacterial and antioxidant potentials of gilaburu (Viburnum opulus L.) fruit extract" // Acta Alimentaria, 2006, v. 35, 487-492]. The presence of the effect of nanocomposites was determined by the width of the zone of inhibition around the wells on the dish with the medium.

To assess the bacteriostatic activity of NA against bacteria, a liquid culture of microorganisms was grown in the dark at 26°C under aerated conditions (80 rpm) in flasks with a nutrient medium. After the addition of the nanocomposites, the optical density of the suspension was measured at 595 nm on a Bio-Rad plate spectrophotometer immediately and after 2, 4, 24, 28, 48, 52, and 72 h of co-incubation.

The experiments were carried out in three independent biological replicates. The data obtained were subjected to statistical processing using the MS Excel statistical processing package and evaluated according to the Mann-Whitney test.

The results showed no bactericidal effect of nanocomposites I - III on the growth of rhizospheric bacterial cultures of A. guillouiae , R. erythropolis and P. oryzihabitans (Fig. 12). It was shown that manganese-containing NCs did not have a bactericidal effect on these bacteria, and no precipitation zones were identified around the wells with composites. It was found that on the first day of conincubation of bacteria with nanocomposites, a decrease in cell growth was noted compared to the control, but later, in the dynamics of observation, this effect was leveled. Thus, it can be concluded that the studied nanocomposites are safe for representatives of soil microflora.

Fig. Fig. 12. Effect of treatment with nanocomposites with manganese concentrations of 0.000625% and 0.00625% ^† in volume on growth (left panel) and biofilm formation (right panel) of A. guillouiae (A), P. erythropolis (B), and P. oryzihabitans (C); 1 - control, 2 - I , 3 - I ^† , 4 - II , 5 - II ^† , 6 - III , 7 - III ^† ; * p ≤ 0.05 and ** p ≤ 0.01 compared with the control according to the Mann-Whitney test.

The results showed that manganese-containing NCs do not lead to any disruption in plant development - plants remain upright, have a developed leaf blade, retain leaf alternation and other characteristic features. At the same time, plants are not infected with a bacterial phytopathogen and stress resistance is stimulated.

Claims (2)

1. Modified nutrient medium for cultivating potato plants based on Murashige-Skoog agar nutrient medium, characterized in that it contains manganese in the form of any of the manganese-containing nanocomposites based on natural polysaccharide arabinogalactan (I), arabinogalactan sulfate (II) or κ-carrageenan ( III) in the amount of 5.49 g/l, determined from the calculation of the required content of elemental manganese in accordance with the Murashige-Skoog prescription and based on the mass fraction of manganese in the nanocomposite. 2. A method for growing potato plants under aseptic conditions using a nutrient medium according to claim 1.

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