Extraction of “Elicitor” and determination of volatile organic substances contained in the elicitor

. In the 21st century, the increased farming activities have harmed both ecosystems and agricultural systems on Earth. This is because of the misuse of mineral fertilizers and toxic plant protection products. To obtain the “elicitor” extract, rhizobacteria were grown in a thermostat for 3 days and then subjected to autoclaving at a temperature of 120°C and 1 atm. for 20 minutes. Azospirillum, Azotobacter, and Pseudomonas are the three most significant growth-promoting bacteria. These organisms influence plant growth regulators, including auxin, gibberellin, and cytokines, in addition to biological nitrogen fixation and soil phosphate solubilization. The research mentions that Azotobacteria create antifungal substances that fight against plant infections, boost seedling viability and germination, and, as a consequence, enhance plant development in general. The result presented that the fifth peak at 10.525 minutes showed the presence of 4(dimethylamino)-α-(P-toluidine) benzylphosphine oxide at 16.0%. The sixth peak at 10.728 minutes contained 19.48% of quinoline-3-carbonitrile, 4-methyl-2-dimethylaminomethylamino, while the seventh peak at 11.177 minutes contained 41.8% of ethanediol % 2 C+bis(phenylhydrazone).


Introduction
In the 21st century, the intensification of agricultural production has had negative consequences for ecosystems and agrosystems on the earth due to the improper use of mineral fertilizers, improper use of plant protection products using toxic substances [1][2][3].
The most important growth-promoting bacteria are Azospirillum, Azotobacter, and Pseudomonas, which affect plant growth regulators, especially auxin, gibberellin, and cytokines, in addition to biological nitrogen fixation and soil phosphate solubilization. It is mentioned in the literature that Azotobacteria produces antifungal compounds that fight against plant diseases, increase the viability and germination of seedlings and, as a result, improve the overall growth of plants [4].
The effectiveness of biopreparations based on bacteria has been reflected in several studies. In particular, it is stated in some sources that the preparations used in medicinal plants have a significant effect on the number of plant leaves, their growth and development, and their quality characteristics [5]. A co-produced formulation of Azospirillum, phosphorus solubilizing bacteria (FEB) and pink pigmented facultative methylotropes were used as bioinoculants in cotton. Plant growth indicators and root growth, yield parameters and fiber quality indicators were determined [6][7][8][9][10].
The technology of obtaining biopreparations is based on liquid phase and solid phase fermentation, and optimization of all stages of these processes is important in the production of biopreparations of competitive quality and in demand. The liquid phase fermentation method uses separation, sedimentation, flotation, vacuum condensation or membrane technologies for the production of biopreparations in liquid and dry form [11].
In recent years, the immobilization of producer strains in carriers with different compositions has become a widely used method. This involves adding or immobilizing the strains onto solid natural sorbents to obtain granular and powdered biological products. Various peat forms, such as Extrasol, Rhizotorfin, and Agrofil, have been used in agriculture to enhance soil fertility. Additionally, these carriers are also used to administer drugs [11][12][13]. The technology of their production is quite simple, does not require large costs, and the nutrient medium allows active reproduction of microorganisms for a long time without losing their viability [14,15].
Today, there are several promising environmentally friendly and harmless means used in plant protection. Among such biological products, there are inducers of resistance to diseases, that is, abiotic and biotic substances -elicitors, which are recognized by the plant and in response to them, the plant activates defense mechanisms, as a result of which the effects of abiotic and biotic stress are relatively reduced [16].

Materials and methods
To obtain the "elicitor" extract, rhizobacteria were grown in a thermostat for 3 days and then subjected to autoclaving at a temperature of 120°C and 1 atm. for 20 minutes (Fig. 1). The resulting cooled autolysate was then filtered [17]. The process of preparing the equivalent material for the production of "elicitor" depends on the type of producer and its physiological and biochemical features [9][10][11][12][13][14][15]. It consists of several stages, including the initial gray form in a test tube, agar cultivation in a test tube, liquid food cultivation in oscillators or special equipment, microorganism ash types accumulation in fermenters, and the production of equivalent material [11][12][13][14][15][16][17].
The "elicitor" extract contained volatile organic substances, which were determined using a specific method. The production process involved the incubation of gray circles in Peptone for 48 hours at 28°C, followed by the transfer of 100 ml of the resulting culture to 500 ml Erlenmeyer flasks containing a fermentation medium (peptone, corn flour, CaCO3, NaCl, glucose, yeast extract, pH-6.8). The cultures were then incubated at 28°C with constant mixing at a speed of 220 rpm for 3 days. After fermentation, the bacterial cells were separated by centrifugation for 20 minutes at 8000 ayl/q using a SIGMA 3-30KS centrifuge. The resulting supernatant was then mixed with a solvent in a volume ratio of 1:10, and 70 mL of organic solvent (n-hexane) was used for extraction [2][3][4][5][6]. Finally, the extract was analyzed using a gas chromatography-mass spectrometry analysis, and 1 μl of the extract was used for the analysis [9][10][11][12][13]17].

Results and discussion
During our research, we required a highly purified biological product called Elisitor, which comprises live microorganisms and inactivated cell biomass, based on purified metabolic products of microorganisms. We found it necessary to obtain a liquid form of the product and developed a technology to do so. To cultivate the microorganisms, we developed a nutrient medium containing peptone (10 g/l), MgSO4 x 7H2O (0.3 g/l), glucose (20 g/l), K2NRO4 (0.4 g/l), NaCl (3.0 g/l), CaCO3 (3.0 g/l), and a pH of 6.8, using distilled water.
The initial batch fermentation process began by introducing the microorganisms into the prepared nutrient medium, and we used bioreactors of varying sizes during different stages of biomass production. To obtain the seed material, we grew up the microbiological equivalent material in the laboratory. We started with a sterile ash in test tubes and transferred it to liquid nutrient medium in Erlenmeyer flasks. We then placed the flasks on shakers at 28 ± 2 0 C and continued the cultivation period for 72 hours. The grown cultures were then used to inoculate the equipment used for seed material preparation.
The main factor in the preparation of seed material was to maintain a moderate growth regime. We transferred the culture fluid to larger volume equipment with pre-sterilized nutrient medium and continued cultivation for 12 hours. We then transferred the suspension to an extractor and obtained the extract, which was directed to the biomass storage tank. After passing through a filter, the filtrate was transferred to a dispenser and packaged. Based on accepted methods for biotechnological processes, we developed a model diagram of technological processes suitable for the microbiological synthesis of Elisitor based on the association of rhizobacteria.
In the analysis of volatile organic compounds in compositions containing elicitors, it has been observed that PGPR elicitors are primarily soluble compounds, but some of them can also be metabolites and volatile organic compounds (VOCs). Plants release these compounds as a response to elicitors, which in turn triggers systematic resistance. In our study, we examined the composition of the biopreparation "Elicitor," which was developed based on rhizobacteria association, using gas chromatography-mass spectrometry. The amount of volatile organic compounds from secondary metabolites in these bioproducts was determined using GX-MS. Our analysis revealed the presence of seven different volatile compounds in the extract of "Elicitor" in n-hexane, as indicated by the number of peaks in the chromatogram (Fig. 2).  (Table 1). Like antibiotics, volatile organic compounds are an important class of secondary metabolites that are effective against pathogenic organisms. That is why the experiences of using them as antibacterial compounds necessary for agriculture, medicine and pharmaceutical industry have been tested in the researches of world scientists. Biologically active volatile compounds produced by Bacillus sp., B. thuringiensis and B. amyloliquefaciens have been modeled as antibacterial agents.

Conclusions
In the case of microbiological protection of plants, the proposed additional measures usually do not involve changing the technology of growing crops, but depend on the treatment of crops and planting crops with bioproducts, that is, they require additional use of technology.
The use of microbiological products -"Elicitor" bioproduct in the system of growing plants in the world is a safe investment solution that guarantees a quick harvest and protects seeds from pests and diseases in stressful conditions and lands with low soil fertility and high levels of therapeutic metabolites-flavonoids.