Isolation and identification of bacteria isolated from cabbage seeds in the Ulyanovsk region

. Bacteriosis caused by the bacterium Xanthomonas campestris is considered one of the most dangerous for cultivated plants. The problem is particularly sharp during favorable agricultural seasons for the development of pathogens. At the same time, effective and modern methods of combating these bacterioses are currently lacking for their effective control. One of the suggested solutions to this problem is the use of phage biopreparations, for which the activity towards "field" cultures of microorganisms is important. In this regard, 22 strains of Xanthomonas campestris bacteria were isolated from 36 samples of seed material. Despite the differences in the seed material, all the isolated strains had typical biological properties for the species and corresponded both to each other and to the reference strains used in work. At the same time, the pathogenicity of isolated microorganisms in relation to cabbage was established in 21 strains. Page layout


Introduction *
Xanthomonas campestris, which is a gram-negative rod-shaped bacterium, is one of the most common pathogens of bacterial plant diseases [1].Subspecies X. campestris pv.campestris (Pammel) Dowson (Xcc), in its turn, causes significant damage to cruciferous crops worldwide, affecting the vascular system of plants [2][3].At the same time, in many cases, the use of standard methods of crop rotation, weed control and, most importantly, the use of bacteriologically pure seeds is sufficient to control the disease [4][5].However, in some cases, especially if it concerns small enterprises, these methods are not effective enough, which increases the relevance of the search for new methods, including such as biocontrol [6][7].
On -time detection of pathogen in seed material before it gets into soil is also relevant, as it allows to prevent infection of both the soil itself and plants from other seed lots.Semiselective nutrient media are usually used to isolate Xcc [8][9].At the same time, the use of phage biopreparations, which can be used both for identification and for bacteriosis control, has great prospects.
In this regard, the purpose of this work was to isolate bacteria belonging to the species Xanthomonas campestris from seed material for the possibility of further work on determining phage activity directly to active pathogens.

Research samples.
The object of the study was 36 samples of cruciferous seeds used for cultivation in the Ulyanovsk region, Russia, obtained in spring of 2022, used by agricultural enterprises of the Ulyanovsk region.To study pathogenicity, cabbage sprouts of varieties F1, Amager 611 and Sibiryachka 60 were used.
Bacterial strains.During the work, 22 strains of Xanthomonas campestris bacteria were obtained.To conduct a comparative biochemical analysis, reference strains of bacteria obtained from the All-Russian Collection of Microorganisms (RCM) were used -B-570, B-610, B-611.
Isolation of microorganisms.Isolation of microorganisms from soil samples was carried out according to the previously offered scheme [10].Seed samples were reduced and suspended in saline solution in a ratio of 1:10 for 2 hours.Then up to 5 dilutions of final suspension were carried out and seeding was done on the YDC medium.Crops were cultivated at +28 °С for 48 hours.The first identification of bacteria was carried out visually by the presence of typical colonies with yellow pigment.Then a pure culture of bacteria was isolated by multiple passage.
Storage of isolated strains.Permanent storage of isolated bacteria was carried out at a temperature of 2-4 °С on a semisolid medium LB (0.3% agar).
Study of pathogenicity.
The pathogenicity of isolated strains in relation to the host plant was assessed by infiltration, for which a bacterial suspension was injected into the intercellular space of the leaves using 26-gauge hypodermic syringe.The syringe needle was inserted under the epidermis from the dorsal side of the leaf.The development of infection was monitored for 1 month with weekly fixation of result in the first half and every 1-2 days in the second half of the month.
Identification of isolated bacteria.It was carried out by coloring isolated cultures by Gram, studying mobility and amylolytic activity.Biochemical tests such as indole formation test, acid formation intensity (reaction with methyl-roth), acetoin production (Voges-Proscauer reaction) and gelatin dilution were performed.The ability of fermentation of glucose, sucrose, lactose and sorbitol was studied.The results were recorded after 48 hours of cultivation.The final evaluation of the results of carbohydrate fermentation was carried out after 120 hours of cultivation.

Results and Discussion
Isolation of microorganisms.
Within the carried out work, 22 bacterial strains with colonies typical of the species Xanthomonas campestris were isolated from 36 seed samples (figure 1).Note that the color of the colonies in some cases differed in hue.At the same time, all colonies had a slime-like consistency characteristic of these bacteria as a result of their formation of xanthan exopolysaccharide.
Study of pathogenicity.
Out of 22 isolated strains studied for pathogenicity, 21 strains were pathogenic toward used cabbage varieties (table 1).Pathogenic strains in the first days of infection caused various symptoms, both characteristic and uncharacteristic for this pathogen.Most strains first caused small grayish spots around the stomata, followed by characteristic V-shaped lesions on the edges of leaves.Subsequently, the leaves drooped.Seven strains caused extensive chlorosis and destruction of mesophilic leaf tissue.17 strains showed yellowing in stomata area with subsequent transformation into necrosis.which showed resistance to only 2 of 22 isolated strains, one of which was a strain that did not show pathogenic properties in this study.
The rate of development of infection signs on cabbage leaves.
Together with the qualitative study of the pathogenicity of the isolated strains of microorganisms, the study of rate of their development of vascular bacteriosis signs in infected plants was carried out.It is worth noting that all isolated bacterial samples visually showed their pathogenic properties not earlier than 16 days after infection of respective plant.
More than 70% of cases of signs of vascular bacteriosis were diagnosed not earlier than on the 20th day, which is associated with the spread and development of pathogen inside the plant.
Identification of isolated bacteria.Summary data on the study of biological properties of isolated microorganisms are shown in the table 2.
It was revealed that isolated cultures were rod-shaped gram-negative bacteria located individually or in short chains (figure 3).Isolated cultures were mobile microorganisms, had amylolytic activity.According to biochemical properties, all isolated strains showed negative reaction with methyl-roth and Voges-Proscauer, diluted gelatin, fermented sucrose and glucose, did not form indole.The studied cultures for the most part did not ferment lactose and sorbitol.The only exceptions were strains CS-8, CS-9, CS-19, CS-22.

Conclusion
Within the framework of this work, 36 samples of cabbage seed material were analyzed, which already at the moment suggests potentially dangerous zones in which, under relevant favorable conditions, the development of bacteriosis and crop loss is possible.22 isolated strains corresponded to present reference strains in their biological properties as much as possible and, which is perhaps important, 21 of the 22 strains demonstrated pathogenic properties towards cabbage samples used in this study.Despite the fact that 1 strain did not show pathogenic properties, but at the same time had similar biological properties with the rest, it was decided to attribute all isolated bacterial samples to the species Xanthomonas campestris pv.campestris.Further analysis of pathogenicity locus will allow us to say with more confidence about the pathogenicity of this strain.
It must be noted that the rate of development of reaction on cabbage leaves was very different between the strains, while most of them caused a visual effect not earlier than 20 days.In part, this phenomenon may be due to quite slow development of plants themselves, which was mainly due to the lack of sunlight in laboratory.Within the framework of this work, the creation of a collection of cellulose-destroying microorganisms was initiated.The conducted studies made it possible to select 39 strains from 14 soil samples, of which 18 were gram-positive bacteria, 21 strains were classified as gram-negative.In the process of studying their cellulolytic properties, it was found that the level of activity between individual strains is very different.5 strains (P 5.1, P 7.3, P 9.3, P17.41, P17.42) with the largest size of the cellulolytic activity zone were selected.The study of some biological properties of isolated microorganisms showed their noticeable difference between each other.However, some similarities have also been noted, such as a lack of activity against tryptophan.

Fig. 3 .
Fig. 3.The rate of development of symptoms of bacteriosis (day).

Table 1 .
Characteristics of pathogenicity of isolated strains.It should be noted that among isolated only 13 strains showed pathogenic properties towards all varieties used in the study.The most receptive was Sibiryachka variety 60,

Table 2 .
Characteristics of the properties of isolated bacteria.