# Characteristic of _Pseudomonas syringae_ pv. _atrofaciens_ Isolated from Weeds of Wheat Field
## 2 Material and Methods  
Bacterial strains used in research. Strains of _Pseudomonas syringae_ pv. _atrofaciens_, which are stored in the collection of microorganisms of the Department of phytopathogenic bacteria of the Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine (NASU), Kyiv were used in the study: _P. syringae_ pv. _atrofaciens_ UCM B-1011 (PDDCC 4394), _P. syringae_ pv. _atrofaciens_ UCM B-1014, and _P. syringae_ pv. _atrofaciens_ 9780. Also, we used the type strain _P. syringae_ pv. _syntgae_ UCM B-1027 (NCPPB 281). Isolation of _P. syringae_ pv. _atrofaciens_. In order to detect the strains of _P. syringae_ pv. _atrofaciens_, wheat fields in the Kyiv region, Ukraine (50\({}^{\circ}\)05\({}^{\prime}\)16.3\({}^{\prime\prime}\) N 30\({}^{\circ}\)02\({}^{\prime}\)56.8\({}^{\prime\prime}\) E) were surveyed 2019 and selected weed plants of _Sonchus arveensis_ L. and _Papaver argemone_ L. with symptoms of bacterial infection were collected. Bacteria isolation was carried out by plating the pieces of plants, bounded with 0.1 mL of sterile tap water, on potato agar. For the bacteria isolation, the parts of plants were selected on the border of healthy and damaged tissues. The morphology and structure of bacterial colonies were studied after 72 h growing on potato agar in Petri dishes [18]. Cell morphology and motility were determined in Gram-stained and \"crushed drop\" preparations, respectively, under a Sigeta MB-201 microscope using a one-day bacterial culture grown in nutrient broth (NB) [5,18]. For the initial selection of the isolates from weeds, the LOPAT test (Levan production, Oxidase, Protopectinase activity on potato, Argin dihydrolase, hypersensitive reaction on Tobacco) was used [18]. Physiological and biochemical characterization. For studying the physiological and biochemical properties of the bacterial isolates from weeds, the test system NEFERMtest24 (MikroLaTEST\({}^{\otimes}\), ErbaLachema, Czech Republic) was used. Oxidase was detected in the cells of the isolated strains by test strips Bacident Oxidase (Merck, Germany). Ferramentative and oxidative glucose metabolism (OF-test) were determined by the breakable microwell plate (OFtest, Erba Lachema). The ability to induce rot on potato was investigated by applying 0.01 mL of bacterial suspension (1 \(\times\) 10\({}^{7}\) colony-forming unit (CFU)/mL) on potato discs [18]. Potato discs were incubated for 24 h at 25 \({}^{\circ}\)C, and then the presence of rot was registered. Pathogenicity tests. To study the virulent properties of isolated strains, artificial inoculation of _S. arveensis_ and _P. argemone_ plants, from which bacterial strains were obtained,was carried out under field conditions and of plants of the spring wheat variety Pecherianka in the greenhouse. Artificial infection of wheat seedlings seven days old was performed by pricking through a drop of bacterial suspension with a concentration of 1 \(\times\) 10\({}^{7}\) CFU/mL. The development of visible symptoms of damage evidenced the bacterium virulence. The results of artificial infection were analyzed according to a 5-point scale in 10 days after the inoculation of bacteria [5]. Highly aggressive strains caused the damage from three to four scores, while weakly aggressive--1-2 scores. Aggressiveness was determined as the average score of disease symptoms in ten inoculated plants. Also, the pathogenic properties of bacteria that were isolated from the weeds were determined by artificial inoculation of the respective plant species from which the strains were isolated. For this purpose, the bacterial suspension containing 1 \(\times\) 10\({}^{7}\) CFU/mL was injected into a leaf by a syringe. Sterile tap water was inoculated into the plant as a control. Aggressiveness was calculated as the average score of the results obtained from 6 experiments (during each of them, five plants were artificially infected).