# Lactic Acid Bacteria as Biocontrol Agents against Potato (_Solanum tuberosum_ L.) Pathogens
## 2 Materials and Methods
### Agar-Plug Diffusion Method for In Vitro Antimicrobial Assay  
The antimicrobial activity of 100 strains of lactic acid bacteria against potato phytopathogens was determined by the agar-plug diffusion method [22]. Lactic acid bacteria were cultured for 48 h at 30 \({}^{\circ}\)C on MRS (Merck, Darmstadt, Germany) agar plates using the pour method to obtain agar plugs, cut with sterile cork bore (\(\Phi\) = 10 mm). Freshly prepared pathogens' suspensions of 100 \(\mu\)L were inoculated onto TSA (Merck, Darmstadt, Germany) for bacteria and PDA (Merck, Darmstadt, Germany) for fungi. Then, lactic acid bacteria agar plugs were transferred onto inoculated plates. Pure MRS agar plugs were used as a control sample. The plates were incubated for 2-5 days (depending on the pathogen) at 25 \({}^{\circ}\)C. After incubation, the width of the phytopathogen growth inhibition zones was measured, excluding the agar plug diameter. The results were expressed as the average of  
\begin{table}
\begin{tabular}{c c} \hline
**Strain** & **Origin** \\ \hline _Alternaria alternata_ & LOCK 408 & Collection of Pure Cultures of Industrial Microorganisms LOCK at the Lodz University of Technology (Lodz, Poland) \\ \hline _Alternaria tenuissima_ DSM 63360 & \\ _Fusarium sambucinum_ DSM 62397 & \\ _Rhizoctonia solanti_ DSM 22843 & German Collection of Microorganisms and Cell Cultures GmbH \\ _Colletotrichum cccodes_ DSM 62126 & (DSMZ, Braunschweig, Germany) \\ _Phoma exigua_ DSM 62040 & \\ _Streptomyces scabiei_ DSM 4077 & \\ \hline _Fusarium oxysporum Z154_ & Plant Breeding and acclimatization Institute (IHAR)—National \\ _Alternaria solanti_ Z184 & Research Institute (Radzików, Poland) \\ \hline _Pectobacterium carotovorum_ PCM 2056 & Polish Collection of Microorganisms of the Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences (Wroclaw, Poland) \\ \hline \end{tabular}
\end{table}
Table 1: Origin of the phytopathogen strains used in the research.  
three independent repetitions for each LAB strain--pathogen combination with a standards deviation value.  
High-Performance Liquid Chromatography (HPLC) for Lactic Acid Bacteria Fermentation by-Products Determination  
Lactic and acetic, propionic, isobutyric and valeric acids were procured from Sigma-Aldrich (St. Louis, MO, USA). Fermentation media with investigating strains were filtered using a 0.45 mm filter before HPLC analysis. HPLC analysis was performed using the Finnigan Surveyor HPLC system (Thermo Fisher Scientific Inc, Waltham, MA, USA) equipped with an autosampler, a refractive index detector (Finnigan Surveyor-RI Plus), a diode array detector (Finnigan Surveyor-PDA Plus) and thermostated column compartment, controlled by ChromQuest 5.0 chromatography software (Thermo Fisher Scientific Inc, Waltham, MA, USA). The investigated compounds were separated on an Aminex HPX87H column (300 \(\times\) 7.8 mm, Bio-Rad, Hercules, CA, USA) protected by a guard column and thermostated at 60 \({}^{\circ}\)C. The separation was carried out by isocratic elution with 0.005 M H\({}_{2}\)SO\({}_{4}\) and a flow rate of 0.6 mL/min; the column temperature was maintained at a constant 60 \({}^{\circ}\)C. The standard of the individual acid was prepared and chromatographed separately to determine each acid's retention time. The calibration curves of individual acids were obtained using five levels of concentration of standard mixtures and three injections per level [23].