Peculiarities of Bacillus Subtilis strains influence on the development of Triticum Aestivum L. in inoculative cultures

Peculiarities of


INTRODUCTION
Sustainable development of crop production and increased crop yields in the face of climate change are possible through the use of highly effective biological products based on active strains of microorganisms.The action of the products promotes the synthesis of natural antidepressants and anti-stress factors of resistance to drought, waterlogging and other adverse environmental conditions in the soil and directly in the plant itself (Verma et al., 2018;Vorobei & Logosha, 2021).Biological products inhibit pathogenic microbiota and form an "induced resistance system" in plants, which prevents the development of diseases from phytopathogenic organisms (Gadzalo et al., 2019).
The creation of inoculants consisting of associations of bacteria with additive and synergistic effects on plants and increasing the resistance of the plant-bacterial system is very promising.In addition, when using different inoculants, it is necessary to calculate the bacterial load on the seed, as well as the technological parameters of the formulations selected for use (Borko et al., 2022).
S.F.Kozar (2021) showed a positive synergistic effect of the introduction of mixed cultures of soil microorganisms into various agrocenoses, which is promising both for physiological indicators of plant growth, activation of the biological system "soil-plant-microbiome", and for the possibility of soil improvement, reducing the pesticide burden on the environment (Borko et al., 2022).Due to targeted selection and optimisation of production technologies for the manufacture of modern microbial preparations, beneficial soil microorganisms -the basis of biological products -acquire higher activity, reproduction rate, resistance to temperature fluctuations, light intensity, etc.A promising scientific and practical area of agricultural microbiology is the formation of multifunctional associations of microorganisms that, when introduced into the metagenome of native soil communities, would return non-commodity crop products to biological cycles, and improve soil properties, increasing their fertility ( Kopylov et al. 2020).The analysis of competitiveness to anthropogenic stress factors creates opportunities for the creation of innovative knowledge-intensive developments for the management of biological processes in agrocenoses.Thus, the selective formation of the diversity of the soil and rhizosphere microbial complex is important for increasing competitiveness and research of complex complexes of plant physiological characteristics.
To ensure the effective introduction of soil microorganisms into crop agrocenoses in the form of various preparative forms, it is necessary to consider several aspects related to the characteristics of the producer strain, the peculiarities of its interaction, the preservation of stability and viability for a long period, as well as the peculiarities and technology of growing a particular crop into the root zone of which beneficial microorganisms are introduced.The methods of seed inoculation used for research purposes are often impossible to use on an industrial scale, and there are significant technical problems with maintaining viable microbial inocula on seeds during seed processing and storage, as noted in several studies by I.K. Kurdish (2018).Other studies show the positive effects of seed inoculation with Bacillus bacteria, such as A.F. Santos et al. (2021), where scientists concluded that an inoculant based on a composition of Bacillus subtilis and B. megaterium is effective for grain yields, as well as for increasing protein content and improving plant fibre digestibility.In other studies, V.F.Guimarães et al. (2021) showed that by using similar inoculants, high efficiency can be achieved in the cultivation of legumes, resulting in productivity statistically higher than the control without inoculation.
Most bacteria (including spore-forming bacteria of the genus Bacillus sp.) released from rhizosphere soil form stable associations, which allows microorganisms to gain several competitive advantages when introduced into the root zone of plants (Mawarda et al., 2020).As a result of the introduction, biological agents are exposed to abiotic and biotic factors that can have both positive and negative effects on bacterial activity.Given the current research on understanding the relationship between rhizobacteria and plants, J. Poveda & F. González-Andrés (2021) and T. Tsotetsi et al. (2022) summarise the current knowledge base on the key metabolites released by both bacteria and plants, their role in the interaction between organisms and responses to various environmental stresses.
According to scientists, bacteria from the Bacillus genus produce secondary metabolites and can be used as natural insecticides for the biocontrol of pathogens through the production of compounds with aseptic activity, synthesis of enzymes that lysate fungal cell walls, and induction of systemic reactions in host plants, and have a positive impact on the soil ecosystem (Saxena et al, 2020), enhance plant growth and development through the production of phytohormones, increase the availability of essential nutrients (nitrogen, phosphorus, iron), and increase ethylene levels through ACC deaminase (Oleńska et al., 2020).
Studies by H. Li et al. (2021) show that biostimulation of maize crops with Bacillus sp.MGW9 strain improves seed germination on saline soils and increases the length of the main root by 7% due to better water uptake, higher chlorophyll, proline, soluble sugar, superoxide dismutase, catalase, peroxidase and ascorbate peroxidase activity, while reducing malondialdehyde content.
In this regard, the search for highly active and new strains of soil microorganisms is relevant for microbiology, biotechnology, soil indication and diagnostics, as well as for screening producers of physiologically active substances.
The research aims to determine the peculiarities of the influence of new strains of B. subtilis on the development of test plants (winter wheat seedlings Triticum aestivum L.) in the case of using inoculated bacterial cultures.

LITERATURE REVIEW
The gram-positive bacterium Bacillus subtilis has a powerful extracellular protein secretion system and does not release any toxic metabolic by-products into the environment.A variety of metabolic processes, and genetic and biochemical variability have led to the widespread use of B. subtilis bacteria in various fields of agriculture, medicine, industry (GRAS status -generally regarded as safe), and in the production of probiotics, immunoreactive factors, enzymes, amino acids, vitamins, etc.It has been proven to increase the decomposition of plant residues (Avdeeva et al., 2016)  A. Shrestha et al. (2014) showed that the adhesion and formation of biofilms by soil bacteria on plant surfaces has a phytopathogenic effect and helps to improve root-soil contact.Thus, the ability of bacteria to form biofilms on plants may be a promising area in agriculture to increase plant productivity.
Bacterisation with soil microorganisms has a positive effect on plant biometrics (height, plant length, root system formation) and dry biomass (Kots et al., 2016).The decrease in inoculation efficiency due to the early application of soil microorganisms can be compensated by using a complex of organic substances (e.g., polysaccharide-protein complex as a stability matrix), which increases the viability of microorganism cells in a resting state.Scientists continue to deepen their knowledge of the regularities of the spatial, taxonomic and functional structure of plant-microbial communities in the root zone of plants and the processes underlying plant-microbial interactions (Krutylo et al., 2017), as well as collect modern monitoring data on the soil microbial biome and carry out comprehensive work on analytical selection of soil microbial strains.
Using metabolomic profiling, they detected and identified molecules produced in roots and root hairs during their infection with rhizobia.Their analysis of soybean root hairs suggested that root hairs synthesise flavonoids, amino acids, fatty acids, carboxylic acids, and various carbohydrates in response to inoculation with the above bacteria, with a high content of trehalose (known as an osmoprotective sugar).To date, several compounds have been identified that are involved in various plant-microbe interactions.For example, g-amino butyric acid, hexahydrohexaoxybenzene-O-methyl, glutamine, and lauric acid, help bacteria overcome the plant's defences during infection.These isoflavonoids and fatty acids appear in free form mainly due to their signalling role in symbiosis, which requires the enhancement of cellular biosynthesis to create structural components necessary to support the development of symbiosis.
For the rational use of the potential of regulatory (signalling) plant-microbe interactions, it is necessary to pay attention not only to the effectiveness of new strains and their compatibility but also to know under what conditions inoculant strains can successfully compete with representatives of soil populations of specific bacteria and colonise the root system of plants.

MATERIALS AND METHODS
The research was conducted at the National University of Life and Environmental Sciences of Ukraine, Department of Soil Science and Soil Protection named after Professor M.K. Shykula.In a model laboratory experiment, the effect of bacterization of Triticum aestivum L. plants with strains H3, H10, H13, H36, H38, H40, H43, and H45, which in previous studies were isolated from the phylloplane and rhizosphere of the root system of winter wheat and typical black soil, identified and assigned to the genus Bacillus sp.B. subtilis (Honchar et al., 2021).
Determination of the optimal load of B. subtilis bacteria on wheat seeds was carried out in a laboratory experiment.Bacterial inoculants in the form of a suspension of B. subtilis cultures were used both in the form of vegetative cells -the beginning of spore formation and complete sporulation (mature culture).B. subtilis strains were cultured on Luria-Bertani (LB) liquid nutrient medium for 72 hours in an orbital shaker incubator at a temperature of at least 28-30°C according to generally accepted methods in microbiology (Tepper et al., 1979;Goldman & Green, 2021).There are treatment options with the following bacterial load: 1) 2.0×10 8 cells/1 seed; 2) 2.0×10 7 cells/1 seed; 3) 2.0×10 6 cells/1 seed; 4) control.Replication was performed four times.
The effectiveness of bacterial inoculants B. subtilis on the growth and development of Triticum aestivum L. was studied in a model experiment.The scheme of the experiment included: 1 -control (without bacterisation); 2-9 (treatment with the appropriate strain of spore-forming bacteria B. subtilis in the form of vegetative cells); 10-17 (treatment with the appropriate strain of B. subtilis bacteria in the form of technological maturity of the crop with complete spore release).In the experiment, the seeds of Triticum aestivum L. were germinated by the roll method.Two layers of moistened filter paper in the form of ribbons measuring 10×55 cm were used, on which seeds were laid out with the embryos down in one line at an interval of 1 cm and Plant and Soil Science, ( 14)3 at a distance of 2.0 cm from the side edges and surface of the ribbons (70 pieces in one row).The seeds laid out on the paper were covered with moistened filter paper and polyethene tapes of the same size and rolled loosely.The wrapped rolls were placed vertically in beakers with B. subtilis culture fluid and placed in a thermostat at 23-250°C (DSTU 4138-200223-250°C (DSTU 4138- ..., 2004).The water level in the beakers was monitored to prevent drying out, and distilled water was added to the beakers with the rolls if necessary (water change every 3-5 days).The germination rate of the test culture (length of roots and seedlings) on day 10 was determined using a conventional centimetre scale.Statistical analysis was performed using Statistica 8.0 software, data were calculated using MS Excel.
Experimental studies of plants (both cultivated and wild), including the collection of plant material, were carried out following the requirements of the Convention on Biological Diversity (1992).

RESULTS AND DISCUSSION
The main principle in the treatment of agricultural plants with microbial preparations with growth-stimulating activity is the need for a detailed study and establishment of the physiologically appropriate time for plant treatment (developmental stage) and determination of the rational inoculation load when applying them.
As a result of biotesting and studying the effect of the culture fluid of new B. subtilis strains on the growth and development of wheat plants, it was found that at dilutions of 1:10, 1:50, 1:100, and 1:500, the stimulating effect of bioagents was observed (Fig. 1).Thus, the lowest stimulatory effect of B. subtilis strains H3, H10, H13, and H36 was observed at a dilution of the culture fluid of 1:500, and the maximum stimulatory effect was observed at a dilution of 1:100.This confirms that the biological efficacy of B. subtilis bacteria is largely due to the production of auxin, cytokinin and gibberellin hormones, and hormonal substances synthesised by microorganisms are extracellular and are present directly in the culture fluid of the strains.
Thus, the study is confirmed by C. Accinelli et al. (2018), where the treatment with beneficial microorganisms B. subtilis increased shoot length (+7%) and root length (+10%) in maize for grain.The method used to achieve the positive effect was the incorporation of the bacteria into a bioplastic-based composition, rather than the use of biofilms or dilutions.G.M. Teixeira et al. (2021) revealed a positive effect of the use of bacterial biostimulation of soybean seeds, which increased germination rate by 15%, root length by 33% and root mass by 27% compared to the control.
The results are generally positive but variable, so more scientific information is needed for different crops and cultivation technologies, considering different beneficial microbes (species and strains) and variable climatic conditions, to understand the impact of seed treatment.
Reports on studies of metabolites of soil microorganisms or physiologically active substances of microbial origin, which are more appropriate for regulating plant growth and development, can be found in studies (Kotsʹ et al., 2016).
The native culture fluid of B. subtilis strains H10, H13, H40, H43 and H45 was found to inhibit the growth of coleoptiles, the rate was lower than the absolute control by 17.0-35.0%.The results obtained indicate the high activity of strains producing auxin-like compounds in the culture fluid and the feasibility of using a diagnostic test for the auxin activity of B. subtilis exometabolites in studies.
In general, the dilution of the culture fluid had a positive effect on the growth of wheat coleoptiles (Fig. 1), which indicates the production of physiologically active compounds in different concentrations by B. subtilis strains (a manifestation of the stimulatory effect).The exogenous supply of active metabolites to plants under the introduction of B. subtilis can also contribute to the optimisation of organogenesis processes and, as a result, increase crop productivity.
It is worth noting that liquid forms of microbial preparations make it possible to apply them with the help of special sprayers during the vegetation of plants in certain phases without additional manipulations.The optimal combination of pasteurisation and treatment of vegetative plants, considering the appropriate rates of preparation and the timing of treatments, is considered extremely important for the development of an effective method of applying microbial preparation.Following scientific, theoretical and practical studies, it has been proven that the signalling interaction between plants and microorganisms is associated with the regulatory functions of plants (especially concerning the rate of reproduction and the number of bacterial cells).Such regulation is very important because the potential rate of reproduction and metabolic activity of bacterial cells is much higher than that of plant cells.Therefore, the interaction of plants and microorganisms is a complex process that is implemented through molecular mechanisms and metabolic integrations (Patyka et al., 2019).
It is known that the morphometric parameters of the growth and development of cultivated plants do not change significantly depending on the method of using microbial preparations, both when treated with the culture fluid of the producer strain and the supernatant of the bacterial culture fluid (O'Callaghan, 2016; Gadzalo et al., 2019).Scientists from around the world have shown that such treatments equally contribute to the improvement of biometric parameters of the culture.In addition, the formation of growth parameters of agricultural plants depends on the rate of application of the microbial preparation.Microbiological regulation of growth processes is an effective means of increasing the resistance of cultivated plants to adverse environmental factors and increasing crop productivity (Pandey et al., 2018) by mobilising the potential capabilities of the plant organism.
The study of O. Lastochkina et al. (2020) confirms the results (Fig. 1) and proves the protective effect of Bacillus subtilis (strain 10-4) against drought stress.The bacterium was applied by biopriming to wheat seeds that were either sensitive (T.aestivum cv.Salavat Yulaev) or tolerant (cv.Ekada 70) to drought conditions during the germination phase.B. subtilis promoted germination and plant growth of 6-day-old seedlings (both length and fresh/dry weight of roots and shoots) under normal growth conditions and rapidly activated specific metabolic adaptations to drought stress conditions by reducing lipid peroxidation, proline content and electrolyte leakage in 21-day-old seedlings.As a result of the studies, the complex parameters of stimulation of B. subtilis bacteria were determined by analysing the germination parameters of the test crop Triticum aestivum L., namely: energy, germination, speed, and friendliness of germination, as well as seedling weight and root weight (based on the cell load per seed).The most positive effect on wheat seed germination was observed for B. subtilis inoculants applied to seeds in mature technological forms (spore culture) and at the rate of 2.0×10 7 cells per seed (Table 1).The positive effect of microbial preparations based on Bacillus subtilis sp. on the biometric parameters of winter wheat plants was also noted in the works of other authors (Liu et al., 2017).Several scientists have described the positive effect of both synthetic growth stimulants ( Calvo et al., 2014) and microbial preparations with growth-stimulating effects (Yadav & Chandra, 2014) on plant growth and development.However, microbial preparations are usually used classically, namely for seed bacterisation.
The laboratory germination rate of winter wheat seeds was more than 92.5% and tended to actively form future seedlings and roots.In general, the germination rate of the test crop met the requirements for the seed.A slight decrease in seed germination under the roll method is compensated by its rapid germination, higher seedling formation index and relatively better growth and development rate.
The germination energy of winter wheat (Triticum aestivum L.) seeds increased by 96.5% when using B. subtilis inoculant bacteria.It should be noted that the use of spore-forming bacteria activates the growth of primary roots of winter wheat plants and, in general, increases the length of the root system (by 6.3-16.5%,respectively, compared to the variant without treatment with the strains).
The treatment of the biotest with inoculated cultures of B. subtilis led to an increase in the raw weight of seedlings by 84.0-109.6%,depending on the experiment variant, compared to the control (Table 2), which indicates the growth-stimulating properties of the new strains.
The data obtained indicate the biological characteristics of the experimental strains of B. subtilis, which are associated with the formation of effective plant-microbe interactions through the exchange of exometabolites.The mechanisms of interaction between bacteria and plants are controlled by both partners and provide them with mutual benefit.This interaction results in the stimulation of plant growth and development and stabilisation of its production process.
It is known that the inoculation of plants with cytokinin-synthesising bacteria stimulates the accumulation of biomass of both shoots and roots (Liu et al., 2017).According to the studies, it was found that the mass of roots using B. subtilis cultures in the form of vegetative cells (B.subtilis strains H3, H10, H13, H36, H43) was 11.8-44.0%lower than in the control.The use of mature spore culture of B. subtilis (B.subtilis H38, H40 and H45) resulted in an increase in root weight by 4.8-11.3%compared to the control variant without bacterisation.Thus, the effect of the studied inoculant strains on the weight of the primary roots of test plants was unequal, and bacterisation of Triticum aestivum L., in general, promotes better development of the root zone of test plants (which may further affect the activation of the adsorption capacity of roots to absorb nutrients from the substrate medium, soil) (Table 2).Summarising the results of the research, it can be noted that new strains of B. subtilis in the form of inoculated cultures had a positive effect on the development of winter wheat Triticum aestivum L., which indicates the production of extracellular hormonal substances by bioagents in different concentrations, the so-called stimulatory effect.The mature technological forms of B. subtilis inoculants accelerated the germination of wheat seeds and activated the growth of primary plant roots and the formation of their root system.The growth-stimulating properties Plant and Soil Science, ( 14)3 of the new B. subtilis strains were also confirmed by the increase in the biomass of both seedlings and plant roots.

CONCLUSIONS
The signalling response of plants to bacterial inoculants in vivo depends on several environmental factors, including plant genotype, which can significantly reduce the efficiency of their practical use.However, pre-sowing seed treatment remains the most affordable and effective agricultural measure today.Since plant-microbial interaction (colonisation of the rhizosphere, plant phylloplane; production of antimicrobial metabolites, physiologically active, phytohormonal substances of auxin, gibberellin, cytokinin nature and vitamins; induction of systemic resistance in the plant, etc.) is considered an important mechanism of biological control of agricultural systems.
Following research results, it is shown that the data obtained at the level of model testing of B. subtilis bioagents on winter wheat (Triticum aestivum L.) plants allow us to identify the positive effect of inoculants associated with the main indicators of plant growth and development and to trace the stimulating effect when using optimal dilutions of the culture fluid.The exogenous supply of active metabolites to plants upon introduction of B. subtilis strains makes it possible to optimise the processes of organogenesis.It was found that at dilutions of 1:10, 1:50, 1:100, 1:500, the stimulating effect of bioagents was observed: from minimal (B.subtilis strains H3, H10, H13, H36 in the case of dilution of the culture fluid 1:500, to the maximum stimulating effect at dilution 1:100).Thus, the biological efficacy of B. subtilis strains is largely due to the production of exometabolites that are concentrated in the culture medium.At the same time, the native culture fluid of B. subtilis strains H10, H13, H40, H43 and H45 inhibits growth processes, which indicates the activation of auxin-like compounds producers in the culture fluid.In the analysis of the effectiveness of plant-microbial interaction in the case of inoculation with different technological cultures of B. subtilis, the expediency of using mature spore cultures of B. subtilis spore cultures (2.0×10 7 cells per seed), which makes it possible to manifest the growth-stimulating properties of new strains, in particular, according to the parameters of the raw weight of seedlings and roots of Triticum aestivum L. Thus, the knowledge about the peculiarities of the influence of new strains of B. subtilis on the development of winter wheat as promising inoculants with the effect of growth stimulation has been expanded.
The search for new producer strains with a protective and stimulating effect and the study of their peculiarities of influence on the plant organism is a promising scientific direction.This will reveal new patterns of spore-forming bacteria and their role in the nutrition of cultivated plants and is also necessary for improving microbial inoculants to increase their manufacturability and efficiency when used in modern crop cultivation technologies.
and the resistance of barley crops to disease development (Kriuchkova & Patyka, 2020).Significant efforts of scientists are aimed at both studying and developing ways to control and change the metabolism, gene expression and protein activity of this bacterium (Cui et al., 2018; Chuiko et al., 2021).Spore-forming bacteria B. subtilis (as well as other representatives of the Bacillus genus -B.megaterium, B. atrophaeus, B. licheniformis, B. amiloliquefaciens, B. pumilus, B. mojavensis, etc.) are among the most sensitive and dynamic components of soil microbial communities, especially under anthropogenic load.Today, research is relevant to study the most active producers of metabolites that have a positive effect on plants -bacteria B. subtilis, B. pumilus, B. brevis, B. megaterium, etc., which promote plant growth through the production of phytohormones, dissolution of inorganic phosphates, synthesis of organic acids, antagonism to phytopathogenic micromycetes, etc.For example, under the influence of phytohormone-producing bacteria, changes in the endogenous hormonal balance of plants can occur (Patyka et al., 2019).Plant and Soil Science, (14)3

Figure 1 .
Figure 1.The activity of physiologically active metabolites of B. subtilis strains and their influence on wheat coleoptiles growth

Table 1 .
Biometric indices of germination of the test culture Triticum aestivum L. under the influence of inoculation cultures of spore-forming bacteria B. subtilis, model experiment

Table 2 .
Influence of B. subtilis inoculation cultures on the wet weight of seedlings and roots of Triticum aestivum L.