# Characteristic of _Pseudomonas syringae_ pv. _atrofaciens_ Isolated from Weeds of Wheat Field
## 2 Material and Methods
The artificial infection of weed plants was evaluated ten days after the inoculation of bacterial suspension due to the five-point scale. The degree of the damage of plants (from zero to five points) was estimated by the size of the necrosis formed: 0--no symptoms of damage; 1--a border around the injection site; 2--small spots of necrosis (up to 5 mm); 3--damage of \(\frac{1}{2}\) part of the leaf; 4--damage of up to 2/3 of the leaf and stems and drying leaves. The ability of _P. syringae_ to induce a hypersensitive reaction on tobacco was determined by the leaf infiltration technique [18]. The suspension of two-day bacterial cultures with a concentration of 1 \(\times\) 10\({}^{7}\) CFU/mL was injected under the leaves' epidermis by a syringe. A cell suspension was prepared in sterile tap water, and it was used as a negative control. The presence of necrosis was observed in a day. The experiment was repeated three times. Serology. For determining the serological properties of the strains, the method of double diffusion in agar was used. The assignment of the strains of _P. syringae_ pv. _atrofaciens_ to a serological group was determined according to the scheme developed in the Department of phytopathogenic bacteria of the Zabolotny Institute of Microbiology and Virology of NASU [19]. Sera were obtained by the immunization of rabbits with live bacteria. The strains of _P. syringae_ that belonged to serogroups II, IV, V, VI were used for immunization [19]. Fatty acids determination. In order to identify the isolated bacteria, the determination of the fatty acids composition of total cellular lipids was carried out. For this purpose, fatty acid methyl ethers were obtained during the methanolysis of whole bacterial cells in a 5% solution of sulfuric acid in methanol [5]. Chromatographic separation and identification of fatty acids were carried out at the Collective Use Center of the D.K. Zabolotny Institute of Microbiology and Virology NASU. The separation of the fatty acid methyl esters was carried out on chromatographic-mass spectrometric system Agilent 6890N/5973 inert. Peaks were identified by comparing their retention times with retention times of standard Bacterial Acid Methyl Ester Mix (26 components), analytical standard, Supelco(r), as well as using an integrated database of NIST 02 mass spectra. The content of individual fatty acids was determined as a percentage of the total peak area. Analysis of fatty acids profiles of _P. syringae_ pv. _atrofaciens_ isolated from weeds and affected by basal glume rot of wheat plants was performed using RStudio (Version 3.5.1) with the basic functions and library \"dendextend\" [20]. For the dendrogram building, the data were normalized, the Euclidean distance was calculated, and the Ward clusterization method was used. RAPD-PCR analysis. RAPD-PCR (Random Amplification of Polymorphic DNA--Polymerase chain reaction) typing was carried out with the primer OPA-13 (5\({}^{\prime}\)-CAGCAC CCAC-3\({}^{\prime}\)) [21]. For DNA extraction, the strains of _P. syringae_ pv. _atrofaciens_ were grown on nutrient broth (NB) at 28 \({}^{\circ}\)C under shaking conditions (160 rpm) for 24 h. The cells were then precipitated by centrifugation on ELMI CM-50 at 8000\(\times\)_g_ for 10 min, resuspended in 0.9% NaCl, and again centrifuged at the same speed [5]. According to the manufacturer's instruction, DNA extraction was carried out with \"DNA sorbent B\" (AmpliSens, Moscow, Russia) kit. The DNA was stored at \(-\)20 \({}^{\circ}\)C.  
Amplification with RAPD primers was performed in a 25 \(\upmu\)L mixture: 200 ng of genomic DNA; 25 pmol of primer; 2.5 Units SynTaQ Polymerase; 0.2 mM of each deoxynucleotide triphosphate; 2.5 \(\upmu\)L of 10\(\times\) PCR buffer (Helicon, Moscow, Russia). The amplification was conducted in Gene ATAQ Controller thermocycler (Pharmacia LKB, Uppsala, Sweden).