## 3 Materials and Methods
### Biological Material  
#### 3.3.1 Bacterial Suspensions  
Three strains of phytopathogenic bacteria, i.e., _Dickeya solani_ IFB0099, _Pectobacterium atrospepticum_ IFB5103, and _Pectobacterium carootovorum_ IFB5118, were utilized in this studies (Table 3). All strains belong to the collection of the Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk (Gdansk, Poland) and were stored at \(-80\)\({}^{\circ}\)C in 40% glycerol.  
Bacterial biomass was obtained from frozen stock and streaked on Trypticase soy agar (TSA) medium. After 24 h of incubation at 28 \({}^{\circ}\)C, a single bacterial colony was taken to inoculate 5.0 mL of Trypticase soy broth (TSB). The 0.5 McF bacterial suspensions were prepared in 0.85% NaCl as follows. Post 24 h incubation at 28 \({}^{\circ}\)C with 150 rpm shaking, the overnight bacterial culture was centrifuged (10 min, 6500 rpm). The obtained pellet was washed twice with sterile 0.85% NaCl. After that, the optical density of the bacterial suspension was set at 0.5 McF (\(\approx\)1-2 \(\times\) 10\({}^{8}\) CFU mL\({}^{-1}\)) by using a densitometer DEN-1B (Biosan, Riga, Latvia). The 0.5 McF suspensions of plant pathogenic bacteria were used to evaluate the antibacterial activity of the APPJ operated under the optimal working parameters.  
\begin{table}
\begin{tabular}{c c c c} \hline
**Run Order** & \(\mathbf{x_{1}}\) & \(\mathbf{x_{2}}\) & \(\mathbf{x_{3}}\) \\ \hline
1 \({}^{\$}\) & 5.0 (0) & 128 (0) & 50 (0) \\
2 & 5.0 (0) & 15 (\(-\)1) & 25 (\(-\)1) \\
3 & 4.5 (\(-\)1) & 128 (0) & 75 (+1) \\
4 & 5.0 (0) & 240 (+1) & 75 (+1) \\
5 & 5.0 (0) & 15 (\(-\)1) & 75 (+1) \\
6 & 4.5 (\(-\)1) & 240 (+1) & 50 (0) \\
7 & 4.5 (\(-\)1) & 128 (0) & 25 (\(-\)1) \\
8 \({}^{\$}\) & 5.0 (0) & 128 (0) & 50 (0) \\
9 \({}^{\$}\) & 5.0 (0) & 128 (0) & 50 (0) \\
10 & 5.5 (+1) & 15 (\(-\)1) & 50 (0) \\
11 & 5.5 (+1) & 128 (0) & 25 (\(-\)1) \\
12 & 4.5 (\(-\)1) & 15 (\(-\)1) & 50 (0) \\
13 & 5.5 (+1) & 128 (0) & 75 (+1) \\
14 & 5.5 (+1) & 240 (+1) & 50 (0) \\
15 & 5.0 (0) & 240 (+1) & 50 (0) \\ \hline \end{tabular} \({}^{\$}\) The center points. \(\mathbf{x_{1}}\)—The He flow rate (in L min\({}^{-1}\)). \(\mathbf{x_{2}}\)—The modulation frequency of the HV AC (in Hz). \(\mathbf{x_{3}}\)—The duty cycle of the HV AC (in %).  
\end{table}
Table 2: The run order of the experimental treatments carried out within the Box–Behnken response surface design multivariate optimization of the He-DBD plasma jet.  
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Bacterial Species** & **Strain Nos.** & \begin{tabular}{c} **Plant of Origin** \\ **(Organ)** \\ \end{tabular} & **Diseases** & \begin{tabular}{c} **Country of** \\ **Isolation** \\ \end{tabular} & **Year of** **Isolation** & **Reference** \\ \hline \multirow{3}{*}{\begin{tabular}{c} _Dickeya_ \\ _solani_ \\ \end{tabular} } & IFB0099 & \multirow{3}{*}{Potato (Tuber)} & Blackleg and & \multirow{3}{*}{Poland} & \multirow{3}{*}{2005} & \multirow{3}{*}{[66]} \\  & (IPO2276; & LMG28824) & & & \\ \hline \multirow{3}{*}{\
\begin{tabular}{c} _Pectobacterium_ \\ _atrospepticum_ \\ \end{tabular} } & IFB5103 & \multirow{3}{*}{Potato (Tuber)} & Blackleg and & \multirow{3}{*}{Canada} & \multirow{3}{*}{1985} & \multirow{3}{*}{SCRI collection} \\  & (SCRI1086) & & & & \\ \hline \multirow{3}{*}{
\begin{tabular}{c} _Pectobacterium_ \\ _carootovorum_ \\ \end{tabular} } & IFB5118 & \multirow{3}{*}{Potato (Tuber)} & \multirow{3}{*}{Soft rot} & USA & \multirow{3}{*}{NA} & \multirow{3}{*}{SCRI collection} \\  & (SCRI1136) & & & & \\ \hline \end{tabular}  
NA—not available.  
\end{table}
Table 3: Characterization of the utilized strains of phytopathogenic bacteria.