## 2 Results
### Antimicrobial Activity of EOs by Agar Spot Test against Pathogen Indicator Strains  
EOs extracted from the hybrid _Origanum vulgare ssp. viridulum \(\times\) Origanum vulgare ssp. hirtum_ showed a relevant antimicrobial activity assayed by an agar spot test, with halos of various sizes (Table 3). In particular, all the pathogenic microorganisms considered were susceptible to the inhibitory activity of CAR1, CAR2 and CAR3, with inhibition halo radii exceeding 7.5 mm, with the exception of _L. monocytogenes_ SA and _Pseudomonas fluorescens_ B13, against which these oils had low activity. Concerning the EOs extracted from _Origanum heracleoticum_, THY3 and THY5 were tested, as they resulted as more divergent within the THY cluster (Figure 1). These two EOs showed a medium activity against _P. putida_ KT2240 and _P. fluorescens_ B13 (radii between 5 and 7.5 mm) and high inhibitory activity against all other pathogenic and alterative microorganisms (Table 3). On the basis of the results of the chemical characterization (Table 1) and antimicrobial activity by spot test (Table 3), CAR1 and THY5 were selected for further experiments, as they had higher carvacrol and thymol content among CAR1, CAR2 and CAR3 and among THY3 and THY5, respectively. Moreover, CAR1 and THY5 showed high antimicrobial activity against _L. monocytogenes_ OH and _S. Typhimurium_ LT2 and were thus chosen as representative of the most frequently reported pathogen related to foodborne diseases. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline  & & & \multicolumn{6}{c}{**Oregon Subspecies/Hybrid**} \\ \cline{4-10} \multicolumn{1}{c}{**Compound/**} & & \multicolumn{3}{c}{**Origanum vulgare**} & & & & & & \\ \multicolumn{1}{c}{**Enantiomer**} & & & & & & & & & & \\ \multicolumn{1}{c}{} & & & & & & & & & & \\ \multicolumn{1}{c}{} & & & & & & & & & & \\ \multicolumn{1}{c}{} & & & & & & & & & & \\ \hline \multicolumn{10}{c}{**(b)**} \\ \hline \multirow{2}{*}{\(\alpha\)-thujene} & n.i. 2 & 950 & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. \\  & n.i. & 952 & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. \\ \hline \multirow{2}{*}{\(\alpha\)-pinene} & S/\(-\) & 984 & 6 & 7 & 6 & 3 & 5 & 4 & 4 & 3 \\  & R/+ & 988 & 94 & 93 & 94 & 97 & 95 & 96 & 96 & 97 \\ \hline \multirow{2}{*}{\(\beta\)-pinene} & R/+ & 1026 & 79 & 79 & 81 & 79 & 79 & 84 & 84 & n.d. \\  & S/\(-\) & 1032 & 21 & 21 & 19 & 21 & 21 & 16 & 16 & n.d. \\ \hline \multirow{2}{*}{\(\alpha\)-phellandrene} & R/\(-\) & 1036 & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. \\  & S/+ & 1039 & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. \\ \hline \multirow{2}{*}{linalool} & R/\(-\) & 1217 & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. \\  & S/+ & 1224 & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. \\ \hline \multirow{2}{*}{terpinene-4-ol} & S/+ & 1300 & 58 & 59 & 58 & 63 & 62 & 61 & 63 & 62 \\  & R/\(-\) & 1304 & 42 & 41 & 42 & 37 & 38 & 39 & 37 & 38 \\ \hline \multirow{2}{*}{\(\alpha\)-terpineol} & R/+ & 1350 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & n.d. \\  & S/\(-\) & 1360 & 100 & 100 & 100 & 100 & 100 & 100 & 100 & n.d. \\ \hline \end{tabular}  
* \({}^{1}\) Linear retention index determined from the Cyclosil-B column stationary phase (30% Heptakis (2,3-di-O-methyl-6-O-t-butyl dimethylsilyl)-6-cyclodextrin in DB-1701). 2 It was not possible to identify the two enantiomers due to the unavailability of standard pure compounds and lack of information from the literature.  
\end{table} 
Table 2: _Cont._In Vitro Antimicrobial Activity of CAR1 and THY5 against L. monocytogenes OH and S. Typhimurium LT2 by Direct Contact Test  
The two EOs CAR1 and THY5 showed similar antimicrobial capacities by a direct contact test against _L. monocytogenes_ OH (Figure 2A). In particular, at a lower EO concentration (0.12%), an approximately 3-log reduction in microbial concentration occurred as compared to samples exposed to 0% EO (C) and to 0% EO plus 0.125% Tween 80 (CTw).