BACTERIA-ANTAGONISTS OF THE AGENTS OF SORYZ BACTERIAL DISEASES

Aim. To isolate and identify bacteria with antagonist properties for biocontrol of the agents of bacterial diseases of soryz ( Sorghum oryzoidum ) and sorghum crops. Methods. The studies were conducted in 2021-2023. Spore-forming bacteria were isolated from the soryz samples, collected in the ﬁ elds of the experimental farm of the Uman National Horticulture University (Cherkasy region, Uman). Lactic acid bacteria were isolated from soryz plants, collected in the private land plot, located between the villages of Teolyn, Vladyslavchyk, Kniazhyky in Monastyryshche community, Uman district, where Pershotravneve hamlet used to be situated. A total of 1,250 samples were analyzed. The experiment had three repeats. Spore-forming and lactic acid bacteria were isolated from the surface of soryz plants on the ﬁ rm ripe stage in summer while isolating phytopathogenic bacteria. The isolates of lactic acid bacteria-antagonists were also isolated from the inner part of winter stubble stalk of soryz, collected from the tilled ﬁ eld. The antagonistic activity of the strains of lactic acid bacteria and spore-forming bacteria, isolated from different ecological niches, to phytopathogens of soryz and sorghum crops was determined in vitro . The strains of Pseudomonas syringae, the agents of soryz bacterial spots, were used as test-cultures: 211141a, 211141, 210341, 21034, and 210521, along with the collection strains of phytopathogens: Pseudomonas syringae 8299, Pseudomonas syringae subsp. syringae UKM B-1021, X. oryzae 8375, Dickeya chrysanthemi 8683, Diskeya chryzanthemi 8683. The antagonistic activity of the extracted isolates of spore-forming and lactic acid bacteria was studied using the method of radial strokes (joint cultivation of the antagonist and the strains under investigation). The bacterial isolates were deemed inactive if the growth delay zone was 0–5 mm (–), from 5 to 10 mm (+) – low activity, 11–20 mm (++) – moderate activity, over 20 mm (+++) – high activity regarding the test-cultures. To check the effect of the isolate-antagonist of phytopathogenic bacteria, arti ﬁ cial infecting was conducted in the ﬁ eld conditions. For this purpose, a diurnal culture of the antagonist was introduced into the stalk of plants in the concentration of 1×10 8 colony-forming units, and 24 h later, a culture of test-strain of the phytopathogen was administered above the previous puncture. The results were evaluated 7–14 days after the arti ﬁ cial infection. The experiment had three repeats. The isolates of bacteria which demonstrated their antagonistic properties regarding the phytopathogenic bacteria were identi ﬁ ed by their morphological properties, Gram staining, catalase test, pro ﬁ le of carbohydrate fermentation and mass-spectrometry (MALDI-TOF – Matrix Assisted Laser Desorption/Ionization) using VITEK MS mass-spectrometer. Results. Thirty-eight spore-forming bacterial iso-lates were extracted from soryz; among these, 21030, 21095, 21040, ASV1, ASV3, B4 demonstrated their antagonistic activity towards the investigated phytopathogenic bacteria. Isolate 21040 showed high antagonistic activity to most test-strains of P. syringae from soryz (the zone of negative culture – 23–30 mm) and lower activity regarding the collection cultures. Isolates B4 and AVS3 demonstrated their selective activity regarding the investigated phytopatho-gens. Twenty isolates of lactic acid bacteria were extracted. Higher antagonistic activity was noted for the isolates of lactic acid bacteria 8/1 and F1 to the strains of P. syr ingae, isolated from soryz and collection cultures. The highest antagonistic activity of isolate 8/1 was noted regarding test-strains of P. syringae 210521 and X. oryzae 8375 (the zone of negative culture – 40–35 mm). In the ﬁ eld conditions, the treatment of sorghum plants with F1 affected the pathological process that developed due to the impact of the phytopathogenic bacteria P. syringae , which led to the reduction in disease symptoms. The taxonomic position of the isolates of bacteria, which seem to be promising for the control of disease agents, was determined. In terms of morphology of cells and colonies, the biochemical pro ﬁ le, and mass-spectrometry MALDI-TOF, the spore-forming isolates 21040 and B4 were identi ﬁ ed as Bacillus subtilis , and ASV3 – as Bacillus vallismortis. The identi ﬁ ed isolates of lactic acid bacteria were Lactobacillus pentosus F1


INTRODUCTION
Agriculture is an important branch of economy.The diseases of crops, caused by bacterial pathogens, result in serious limitations to agricultural production and lead to considerable annual losses on the global scale (Sundin et al, 2016).Traditional agriculture uses chemical fertilizers and pesticides to enhance performance and to control disease agents and pests.However, the increase in their application impacts the ecological equilibrium and safety of food products and is the main factor of environmental pollution (Ra man et al, 2022).Th erefore, as an alternative to chemical preparations, researchers pay great attention to the elaboration of ecologic methods of protection against crop disease agents, using microbial preparations, based on different species of microorganisms and their metabolites (Bergsma et al, 2022).
Among bacteria-antagonists of plant disease agents, special attention is paid to the spore-forming bacteria of genus Bacillus.The se bacteria are characterized with high viability in the environment, they are tolerant to anthropogenic effects and are technological both in production and application.The bacteria of Bacillus group are microorganisms, inhabiting a number of various environments.They are well-known producers of a wide spectrum of antagonistic compounds of different structure and cover from 5 to 8 % of the total genome, designated for the biosynthesis of secondary metabolites.The strains of Bacillus genus demonstrate their ability to conduct biocontrol mainly via the inhibition of the growth of plant pathogens and triggering of systemic resistance in plants and competing with plant pathogens for ecologic niches (Fira et al, 2018).
The foundation of biopreparations for the biocontrol of disease agents consists of the representatives of rhizosphere microorganisms of genus Bacillus, which demonstrate high antagonistic activity regarding the phytopathogenic microorganisms due to the synthesis of exo metabolites of different nature -antibiotics, bacterial toxins, volatile organic substances, biosurfactants (Compant et al, 2005), ph ytohormones and vita-mins (Araújo et al, 2005;Chen et al, 2010), which have a positive impact on the growth and development of plants and protect them from phytopathogenic microorganisms.
The Bacillus bacteria colonize the rhizosphere of plants, and the root exometabolites, released by plants, serve as nutrients for bacteria (Saharan et al, 2011).
Seve ral researchers demonstrated high antagonistic activity of Bacillus bacteria to the agents of fungal and bacterial diseases of plants (Lapa et al, 2005;Drahovoz et al, 2014;Hrabova et al, 2015).It was demonstrated that Bacillus bacteria inhibit the development of the infection, caused by Clavibacter michiganensis subsp.michiganensis on tomatoes.The presowing treatment of the seeds with the suspensions of Baci llus subtilis IMV B-7023 and Bacillus pumilus 3 enhanced the resistance of plants to bacterial cancer, which could have occurred due to the synthesis of biologically active substances with anti microbial properties by these bacteria.A considerable stimulating effect on the growth and development of tomatoes was ensured by the strain of B. subtilis IMV B-7023, which is a component of bacterial preparations for plant production (Roi et al, 2012).
The antagonistic activity is also notable for saprotrophic strains of Bacillus, isolated from the surface of green leaves of root artichoke.It was determined that the strain of Bacillus subtilis UzNU-18 demonstrated high antagonistic activity to phytopathogenic fungi and phytopathogenic bacteria (Davr anov et al, 2019).
A relevant role in the study of antagonist microbes is also attributed to lactic acid bacteria.The distribution of lactic acid bacteria (LAB) in soil is minimal and not dominant, but they are considered to be promising candidates for agriculture, promote plant health, and soil fertility.Thus, they are closely considered for the purposes of sustainable agriculture.LAB metabolites promote the growth of plants and stimulate the growth of shoots and roots.As fertilizers, they may promote biod ecomposition, increase the content of organic substances in soil and produce metabolites of organic acids and Lac tobacillus sakei 8/1.Conclusions.In addition to phytopathogenic bacteria, from soryz plants we isolated the strains of spore-forming bacteria Bacillus subtilis 21040, B4, Bacillus vallismortis AVS3 and such lactic acid bacteria as Lactiplantibacillus pentosus and Lactobacillus sakei 8/1 (Latilactobacillus sakei 8/1), promising for the elaboration of methods for the biocontrol of the agents of bacterial diseases.
According to scientifi c literature, plants interact with many groups of lactic acid microorganisms, including Enterococcus, Lactobacillus, Lactococcus; the most notable among them being the coccal forms of Streptococcus lactis, S. cremoris, S. durans, S. faecalis.As for rod-like forms, the most common are Lactobacillus plantarum and L. brevis, which are found in the rhizosphere, on the surface, fruit, and fl owers of plants (Mundt et al, 1968;Rodríguez R et al, 2019).
Wide spreading on fruit (mango), vegetables (tomatoes, squash, potatoes, eggplants), grains (rice, sorghum) is noted for Lactobacillus plantarum, which is found on the surface of plants and fruit (Daranas N et al, 2019).
The groups of LAB on plants may also manifest themselves as growth stimulators (Merlich et al, 2016) and antagonists of phytopathogenic microorganisms (Roi et al, 2012;Steglinska et al, 2022).
The studies of some scientists (Visser et al, 1986;Merlich et al, 2016) showed the antagonistic activity of the representative of LAB, Lactobacillus plantarum, isolated from plant surfaces, towards the test strains of phytopathogenic bacteria of genera Xanthomonas, Pectobacterium, Pseudomonas and Rhizobium (Visser et al, 1986;Merlich et al, 2016).Some authors remarked on the high antagonistic activity of L. plantarum in the MRS medium to both potentially pathogenic and phytopathogenic microorganisms.The investigated strains of L. plantarum produce complexes of metabolites with antagonistic activity.It was shown that lactic acid and hydrogen peroxide are the main factors of the antagonistic effect of most L. plantarum strains, investigated in this work (Harmasheva et al, 2015).There is also a discussion on the role of LAB in maintaining the health of soil and enhancing the performance of crops (Raman et al, 2022;Lamont et al, 2017).The authors found that LAB can improve the soil structure, increase the availability of nutrients, and inhibit the growth of pathogenic fungi.They also determined that bacteria can promote the growth of plants (Abhyankar et al, 2022).

MATERIALS AND METHODS
The stud ies were conducted in 2021-2023.Sporeforming bacteria were isolated from the soryz samples, collected in the fi elds of the experimental farm of the Uman National Horticulture University (Cherkasy region, Uman).Lactic acid bacteria were isolated from soryz plants, collected in the private land plot, located between the villages of Teolyn, Vladyslavchyk, Kniazhyky in Monastyryshche community, Uman district, where Pershotravneve hamlet used to be situated.A total of 1,250 samples were analyzed.The experiment had three repeats.
The spore-forming bacteria were isolated from the surface of soryz plants (Sorghum oryzium) -a leaf, a stalk, seeds in the fi rm ripe stage in summer, during the release of phytopathogenic strains, causing bacterial infections (Patyka et al, 2017).To isolate potentially active antagonists of phytopathogenic bacteria, which can be distributed by insects in soryz fi elds, we took pollen samples from soryz and isolated lactic acid bacteria from them by the method described in (Belhadj H et al, 2014).
To isolate lactic acid bacteria from soryz, the samples were taken from two plots of different fi elds, one -with organic fertilizer (cow dung), and the other -without any fertilizers.The isolates of lactic acid bacteria-antagonists were also extracted from the winter stubble stalk of soryz, collected from the tilled fi eld.The inner part of stalk pulp was taken.The pulp pieces were extracted from the upper and lower parts, close to the root.The pulp was pestled in the mortar with some sterile water, the homogenates were incubated at 30°C for 24-72 h.After the incubation, the homogenates were applied to the slides with Lactobakagar.The dishes were incubated at 30 ºC for 24-72 h in microaerophilic conditions.The colonies, notable for LAB, were isolated and sown to separate specifi c isolates.
The antagonistic activity of the strains of LAB and spore-forming bacteria, isolated from different ecological niches, to phytopathogens of soryz and sorghum crops was determined in vitro.The following bacterial cultures, the agents of soryz bacterial spots, were used as test-cultures: 211141a, 211141, 210341, 21034 and 210521, along with the collection strains of phytopathogens: Pseudomonas syringae 8299, Pseudomonas syringae subsp.syringae UKM B-1021, X. oryzae 8375, Diсkeya chrysanthemi 8683.
The antagonistic activity was studied using the method of radial strokes (joint cultivation of the antagonist and the strains under investigation) by the well-known method (Patyka et al, 2017).
To check the impact of the antagonists of LAB on sorghum crops in the fi eld conditions, the culture of LAB F1, cultivated for 48 h/37 ºC on solid culture medium MRS in facultative anaerobic bacteria, was diluted in sterile running water to the concentration of 1×10 8 colon y-forming units (CFU) and administered into the stalk of grain sorghum on the stage of panicle earing.The experiment was repeated three times.
The antagonism of LAB to sorghum plants was checked using the modifi ed method of (Daranas, 2019).For the study purposes, we selected the strains of test culture of phytopathogenic bacteria, to which the antagonist demonstrated different inhibiting effects.A diurnal culture of the antagonist was administered into the plant stalk in the concentration of 1 × 10 8 CFU, and 24 h later, a culture of the phytopathogen strain was introduced above the previous injection site.The results were evaluated 7-14 days after the artifi cial infection.
The isolates of bacteria which demonstrated their antagonistic properties regarding the phytopathogenic bacteria were identifi ed by their morphological properties, Gram staining, catalase test, and profi le of carbohydrate fermentation (Bucka-Kolendo et al., 2021).The taxonomic position of the extracted bacterial isolates was confi rmed by mass-spectrometry (MALDI-TOF -Matrix Assisted Laser Desorption/Ionization) using VITEK MS mass-spectrometer (Mellmann et al, 2008;Kačániová et al, 2019;Tsuchida et al, 2020;Bucka-Kolendo et al, 2021).For this purpose, a calibration curve of the strain of Escherichia coli ATCC 8739, cultivated for 24 h, was prepared.A thin layer of cells of this strain was applied to the target calibration well and 1 μl MATRIX was added to each target calibration well.Then pure cultures of the investigated bacteria were prepared for 18-24 h, and the colony of 1 μl was applied to the target well.The introduced culture was immediately administered 1 μl MATRIX into the center of the target well.The target well was kept until it went completely dry and MATRIX crystals were formed on it.The slide was placed into the device which was then switched on using the corresponding software till the process completion.The obtained results are refl ected in the form of a system on the chart, a row of lines -a spectrum, which corresponds to different molecules that separated from the sample.The identifi cation of microorganisms is based on obtaining a total mass-spectrum of proteins in the range of 1,000-10,000 Da and the bioinformational comparison of the obtained spectrum against the database of reference spectra.If the spectrum of the investigated sample coincides with one of the spectra in the database, the system identifi es the sample.If the mass-spectrum of the investigated bacterial strain coincides with one of the spectra in the database of reference bacterial species, the system identifi es it.The range of the probability for accurate identifi cation in percentage is from to 99 (Tsuchida et al, 2020).

RESULTS
To control th e agents of bacteriosis of soryz and sorghum crops, we investigated the bacteria-antagonists, isolated from the surface of soryz plants in summer and from winter stubble of soryz plants, and determined the antagonistic activity of spore-forming strains and lactic acid bacteria.
We found that isolates 21030, 21095, 21040, ASV1, ASV3, B4, isolated from soryz, which were similar to bacteria of genus Bacillus in their morphology, demonstrated antagonistic activity to the investigated phytopathogenic bacteria from soryz and collection strains (Table 1, Fig. 1).
High antagonistic activity was manifested by the isolates of lactic acid bacteria, isolated from the residues of soryz plants which were either on the soil surface or in soil at the depth of 1-5 cm.The prevailing part of plant residues were parts of roots with the stalk of 15-20 cm (Fig. 2, b, c), the inner part of stem pulp was wet (Fig. 1, а).The pulp pieces were extracted from the upper and lower parts of the stalk closer to the root, after cutting off the upper part of the stalk (Fig. 2, b).
Higher antagonistic a ctivity was manifested by the extracted isolates of lactic acid bacteria 8/1 and F1 (Table 2).
The highest antagonistic activity of isolate 8/1 was noted regarding test strains of P. syringae 210521 and X. oryzae 8375 (zone of negative culture -40-35 mm).
The isolate 8/1 demonstrated high antagonistic activity regarding strains P. syringae 211141a (30 mm), 211141 (20 mm), 210341 (30 mm), and was moderately active regarding Dickeya chrysanthemi 8683 (11 mm).High antagonistic activity was registered regarding the collection strains of X. oryzae 8375 and P. syringae subsp.syringae UKM B-1021.We found the effect of isolate F1 on the pathological process, caused by the strains of phytopathogenic bacteria P. syringae (Fig. 3).The results of artifi cial inoculation are a decrease in or the absence of infection symptoms.
However, the treatment of grain sorghum plants with the isolate F1 did not affect the pathological process in the stalk of a plant, which gets developed under the effect of phytopathogenic bacterium X. oryzae 8375.
The re sults of the treatment of sorghum plants correlate with the data, obtained while investigating the antagonism of LAB regarding the test cultures of phytopathogenic bacteria in vivo.
Since the extracted isolates of bacteria-antagonists are perspective for the elaboration of biopreparations for the control of phytopathogenic bacteria, we conducted their identifi cation.
By their morphology, the isolates taken from the surface of soryz plants, which demonstrate antibacte-rial activity towards phytopathogenic bacteria, are si-milar to bacteria of genus Bacillus.These are large, branched, irregular in form, white colonies; the cells are gram-positive, rod-like, and spore-forming (Fig. 4).
The biochemical profi le of the extracted isolates with the antagonistic activity to the phytopathogenic bacteria was evaluated on the microbiological analyzer VI-TEK® 2 Compact (Table 3).By the obtained properties, the spore-forming isolates B4, 21040, and ASV3 had a biochemical profi le, typical for bacteria Bacillus subtilis.
According to the results of MALDI-TOF, the massspectra of isolates B4 and 21040 were identical and corresponded to Bacillus subtilis (Fig. 5, Table 4).
Bacillus is a large genus of gram-positive bacteria, widespread in soil and other media.It is notable for good resistance and ability to form highly stable spores which can survive in severe conditions, including extreme temperatures, dry conditions, etc. (Tu et al, 2021).Bacillus subtilis bacteria inhibit the reproduction of phytopathogenic bacteria by the products of its activity and stimulate the immunity of plants to diseases, which decreases the possibility of recurrent infections (Araújo et al, 2005;Chen et al, 2010).
Isolate ASV3 had a biochemical profi le, typical for B. vallismortis.In scientifi c literature, B. vallismortis is described as an antagonist with antifungal activity (Degrassi et al, 2020).
Thus, the extracted spore-forming isolates with high antagonistic activity regarding the phytopathogens of sorghum crops were identifi ed as Bacillus subtilis 21040, Bacillus subtilis B4 and Bacillus vallismortis ASV3.
The isolates of bacteria F1 and 8/1, extracted from the remains of soryz stalk in winter, which manifested antagonistic properties to phytopathogenic bacteria, had a rod-like form (Fig. 5).By their morphological and biochemical properties, they were similar to bacteria of genus Lactobacillus (Table 5).
According to the scientifi c literature, the antagonistic activity was noted for the representative of lactic acid bacteria Lactobacillus plantarum from plant surfaces to the test-strains of phytopathogens Xanthomonas campestris, Erwinia carotovora (Merlich et al, 2016).
The studies on DNA-DNA hybridization demonstrated that Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum form a closely related group, known as "a group of Lactobacillus plantarum" of gene Rec Lb. plantarum and Lb.pentosus Hind II Lactobacillus pentosus (Torriani S et al., 2001).Fatty acids of L. pentosus demonstrate their antifungal activity regarding fungal pathogens (Lipinska, 2014;Raman et al, 2022).
According to the results of our study, isolate 8/1 had a biochemical profi le, typical for Lactobacillus sakei.The probability (accuracy) of identifi cation was 99 %.
Lactobacillus sakei (Latilactobacillus sakei) is a gram-positive rod-like anaerobic bacterium.The scientifi c literature states that this species decreases the number of viable microorganisms of Enterobacteriaceae and Pseudomonas sp.families, and in case of long-term storing, L. sakei in the medium with pathogens manifests a more inhibiting effect (Zhang, 2018).
The most frequently mentioned tomato plants were L. plantarum, L. fermenti, L. brevis, whereas L. casei, L. viridescens, L. cellobiosus, L. salivarius and L. buchneri were obtained from a small number of samples.Wide yet sporadic distribution of lactic acid bacteria in small amounts demonstrates that these organisms do not usually fl ourish on plant surfaces (Mundt et al, 1968).Lactobacillus pentosus Lactobacillus sakei 99.9 99.9 Table 6.The accuracy of identifi cation of bacterial isolates using Vitek MS-DS

DISCUSSION
Bacterial diseases are known to cause considerable damage to agriculture, decreasing the quality and quantity of the cultivated products.The same is true for the agents of bacterial diseases of soryz (Sorghum oryzoidum).Therefore, in our studies, we used the strains of the agent of soryz bacterial spots and sorghum crops as test-culture in the search for microbes-antagonists.
Unreasonable doses of mineral fertilizers, the use of chemical means to control plant diseases and the failure to comply with the technology of their application bring harm to the environment (Raman et al, 2022).Thus, scientists search for ecological methods of protection from the agents of crop diseases, which could be found in microbes-antagonists for the elaboration of biopreparations on their basis (Dragovoz et al, 2014;Hrabova et al, 2015;Davranov et al, 2019;Bergsma et al, 2022).
In the scientifi c literature, the main attention is paid to spore-forming and LAB with antagonistic properties regarding the agents of bacterial diseases of fungal and bacterial nature (Lapa et al, 2005;Merlich et al, 2016;Steglinska et al, 2022).Therefore, while isolating the agents of soryz bacterial diseases, we paid attention to the colonies of spore-forming and lactic acid bacteria, selected for the studies.We found that some sporeforming and lactic acid isolates demonstrated their antagonistic activity both to the strains of Pseudomonas syringae, isolated from soryz, and to the collection culture of genera Pseudomonas, Xanthomonas, Dickeya.The spore-forming isolates of bacteria (21095, 21030, 21040, B4, AVS3) and lactic acid bacteria (F1, 8/1) were heterogeneous by their antagonistic effect regarding the strains of phytopathogens under our investigation.The highest antibacterial activity was found in the isolates 21040, B4, AVS3, F1, 8/1.The investigated isolates of bacteria-antagonists demonstrated higher antibacterial activity regarding strains P. syringae, isolated from soryz bacterial spots than to the collection strains of P. syringae 8299, P. syringae subsp.syringae UKM B-1021, X. oryza e8375, D. chrysanthemi 8683.Similar results were described by other researchers who registered the antagonistic activity of strains of Bacillus subtilis IMV B-7023 and Bacillus pumilus 3 to phytopathogenic bacteria (Roi et al, 2012).These strains differed in their antagonistic effect regarding Clavibacter michiganensis subsp.michiganensisі and demonstrated a higher stimulating impact on the growth and development of tomatoes during the treatment of seeds with the suspension of B. subtilis IMV B-7023 (Roi et al, 2012).
The strains of LAB L. plantarum were heterogeneous by their spectra of antagonistic activity and differed in terms of potentially pathogenic and phytopathogenic microorganisms (Harmasheva et al, 2015).
Although the rhizosphere bacteria of genus Bacillus, which have antagonistic properties regarding the phytopathogenic microorganisms, are considered to be the main component of biopreparations for the protection of plants from disease agents (Compant et al, 2005;Araújo et al, 2005), we isolated the spore-forming bacteria from the surface of plants.Some authors also described that saprophytic strains of genus Bacillus, isolated from the surface of plants of root artichoke demonstrated high antagonistic activity to phytopathogenic fungi (Fusarium oxysporum, Fusarium culmorum, Fusarium solani, Rhizoctonia solani, Phytophtora capsici, Alteranria alternata) and phytopathogenic bacteria (Pseudomonas syringae, Erwinia carоtovora, Xanthomonas beticola) (Davranov et al, 2019).
The scientifi c literature describes the antagonistic activity of the representative of LAB, Lactobacillus plantarum, isolated from plant surfaces regarding teststrains of Xanthomonas campestris, Pectobacterium carotovora, Pseudomonas syringae and Rhizobium radiobacter (Visser et al, 1986;Merlich et al, 2016).The level of antagonism of L. plantarum regarding phytopathogens differed depending on the strain of lactobacilla, its origin and the strain of phytopathogen (Merlich et al, 2016).
We found that in the fi eld conditions, the isolate of LAB F1, which had high antibacterial activity regarding the phytopathogenic bacteria, impacted the pathological process that developed at the effect of phytopathogenic bacteria P. syringae, which led to the reduction in infection symptoms or even their complete absence.
According to the studied biological properties, the spore-forming isolates B4, 21040, and ASV3 had a biochemical profi le, typical for bacteria Bacillus subtilis.
The mass spectrometry MALDI-TOF demonstrated that isolates B4 and 21040 were identical and corresponded to the species of Bacillus subtilis.Isolate ASV3 had a biochemical profi le, typical for B. vallismortis, which is most closely related to Bacillus sub-tilis.According to the data of Roberts et al, (1996), B. vallismortis can be distinguished from B. subtilis only by its differences in the composition of fatty acids in the entire cell.
In the scientifi c literature, B. vallismortis is described as an antagonist with antifungal activity, which may demonstrate its potential as an alternative resource for the biocontrol of plant diseases, but the antagonistic activity of this species regarding phytopathogenic bacteria has not been studied suffi ciently (Degrassi et al, 2020).
L. сasei was often isolated from the surface of tomato plants (Mundt et al, 1968).We isolated L. pentosus bacteria with the antagonistic properties regarding the agents of bacterial diseases, which, according to the data of some authors, was insuffi ciently studied as an antagonist of phytopathogenic bacteria (Lipinska, 2014;Raman et al, 2022).
Since the late 2000s, the technology MALDI-TOF has been used in medicine for rapid identifi cation of bacterial species (Mellmann et al, 2008), and the obtained results are diagnostically relevant.The advantages of this technology are convenience, rapidness, and accuracy in comparison with traditional biochemical methods (Tsuchida et al, 2020).MALDI-TOFMS analysis is a method, widely used for the identifi cation of microbial species, since it ensures rapid obtaining of reliable results (Kačániová et al, 2019).The researchers demonstrated that thirteen out of fi fteen strains of Pseudomonas, identifi ed using MALDI-TOFMS analysis, were additionally confi rmed by the sequencing of gene 16S rDNA (Kačániová et al, 2019).It was shown that MALDI-TOFMS is more accurate for the identification of some bacterial species than partial sequencing of gene 16S rRNA (Mellmann et al, 2008).
Twenty isolates of LAB were extracted.We determined high antagonistic activity of the isolates of LAB F1 and 8/1, extracted from the remains of soryz plants which were either on the surface or in soil, at the depth of 1-5 cm.The prevailing majority of the remains were parts of roots with the stalk of 15-20 cm.It was determined that the isolates of LAB demonstrated higher antibacterial activity to the investigated test-cultures of phytopathogenic bacteria than spore-forming isolates.The antagonistic activity of isolate F1 was confi rmed in the fi eld conditions.Under artifi cial infection, it reduced the symptoms of the pathological process, caused by the phytopathogenic bacteria P. syringae.
Based on the investigated biological properties, the isolates of spore-forming bacteria, extracted from soryz plants, were identifi ed as Bacillus subtilis 21040, B. subtilis B4, Bacillus vallismortis AVS3.The isolates of LAB were identifi ed as Lactiplantibacillus pentosus and Lactobacillus sakei 8/1 (Latilactobacillus sakei).The isolated bacteria-antagonists are recommended for the biocontrol of the agents of bacterial diseases.
To identify the antagonists of genera Lactobacillus and Bacillus, we used MALDI-TOF mass-spectrometry which ensures the identifi cation of microorganisms in one day with considerably high accuracy.The versatility of this method is much more accurate than traditional biochemical methods.
Adherence to ethical principles.All the experimental results, presented in this article, were obtained without the use of any animals.Confl ict of interests.The authors declare the absence of any confl icts of interests.Financing.This study was not fi nanced by any specifi c grant from fi nancing institutions in the state, commercial or non-commercial sectors.

Fig. 2 .
Fig. 2. A stalk, from which the antagonists were isolated in winter: a -the inner part, containing moisture, b -a cross-cut of the stalk; c -a part of the stalk, from which samples were extracted

Table 1 .
The antagonistic activity of spore-forming isolates towards the agents of bacterial diseases

Table 2 .
The antagonistic effect of lactic acid bacteria regarding the agents of bacterioses

Table 4 .
The accuracy of identifi cation of bacterial isolates using Vitek MS-DS

Table 5 .
Morphological and biochemical characteristics of lactic acid bacteria Note. "+" -a positive result; "-" a negative result.