Evaluation of Antiphytoviral and Antibacterial Activity of Essential Oil and Hydrosol Extracts from Five Veronica Species

: Agricultural production without pollution is possible using bioactive plant compounds, which include essential oils as important substances of plant origin. The aim of this study was to evaluate the antiphytoviral and antibacterial potentials of lipid (essential oil, EO) and water (hydrosol, HY) extracts from ﬁve Veronica species (Plantaginaceae) obtained by Clevenger hydrodistillation (HD) and microwave-assisted extraction (MAE), with analysis by gas chromatography coupled with mass spectrometry. The antiphytoviral activities of both extracts were tested on local host plants infected with tobacco mosaic virus (TMV). The antibacterial potential was tested against ten strains of opportunistic pathogens using the broth microdilution test. Species V. chamaedrys EO-MAE extract, V. arvensis EO from both extractions and V. montana, V. serpyllifolia , and V. persica EO-HD extracts were more effective in inhibiting TMV infection. Furthermore, HY-HD extracts of V. arvensis , V. chamaedrys and V. persica showed signiﬁcant antiphytoviral activity. HY fractions had no effect on bacterial growth, regardless of the Veronica species tested, likely due to the fact that the maximum concentrations of the HY fractions tested in this study were low (1.83 and 2.91 mg/mL). EOs showed signiﬁcant antibacterial activity independent of the extraction method. Notably, V. chamaedrys EO-MAE fraction, showed signiﬁcantly better activity against Listeria monocytogenes and Enterococcus faecalis . Also, the EO-HD fraction of V. arvensis showed slightly better antibacterial activity. By combining extracts and using different extraction methods, valuable bioproducts can be obtained from the investigated Veronica species for safe use in agricultural production and food conservation.


Introduction
The results of phytochemical studies on a number of higher plants indicate that plants are endowed with pesticidal properties that can be exploited for use in agriculture and related fields. The need to use plant-based products arises from the fact that synthetic pesticides can be harmful to humans and to the entire ecosystem due to their high toxicity and persistence [1][2][3].
According to previous studies, most plant species with biopesticide activity belong to the Fabaceae and Lamiaceae families, followed by Apocynaceae, Cucurbitaceae, Euphorbiaceae, and Plantaginaceae [4]. The subjects of this study are representatives of the family Plantaginaceae, i.e., five species of the genus Veronica: V. arvensis L. (Corn speedwell), V. chamaedrys L. (Germander speedwell), V. montana L. (Wood speedwell), V. serpyllifolia L. (Thyme-leaved speedwell) and V. persica Poir. (Persian speedwell). These plants were selected for this study because the genus Veronica is the largest in this family and it is widely distributed [5][6][7], making it an easily accessible plant material for analysis and use. All five species studied belong to the medicinal plants, and previous studies on their pharmacological activity focused on iridoids, flavonoids, and saponins [7][8][9]. The activity of other specialized metabolites in the Veronica species, such as the free volatile compounds (FVCs), has been much less studied and has not been systematically investigated [10][11][12]. Our previous research on five Veronica species that are the subject of this study focused on the extraction and identification of FVCs isolated by two techniques, hydrodistillation in a Clevenger apparatus (HD) and microwave-assisted extraction (MAE). Each extract obtained consists of two phases: a lipophilic phase (essential oil, EO) and an aqueous phase (hydrosols, HY) [13][14][15]. The hydrosol part of the extract is often wrongly classified as wastewater [16,17], although it contains very valuable bioactive components. These isolates are natural substances of plant origin and are therefore among the potential biocontrol products. This was confirmed by Raveau et al. in a review article that classifies biocontrol products into four main classes: macroorganisms, microorganisms, semiochemical products, and natural products derived from plants, algae, microorganisms, animals, or minerals [18]. Substances of plant origin were classified as non-living biocontrol natural products by Stenberg et al. [19]. They emphasized that the division into living and nonliving biological substances is important to maintain scientific clarity and the integrity of biological control. These authors define biological control as the use of living organisms, including viruses, to control pests and pathogens that directly or indirectly affect human welfare [19]. It is precisely because of the impact on human health and the risk to the environment that biocontrol products, especially EOs, are of great scientific interest, as they are products of biological origin that are considered more ecological and alternative solutions compared to synthetic pesticides [20]. Among plant diseases, viral infections are of particular concern because of the lack of effective means of virus control in crop protection. Treatments with EOs and HYs of five selected Veronica species were conducted to evaluate the activity of their volatiles against tobacco mosaic virus (TMV) infections. TMV is a positive-strand RNA virus and one of the viral pathogens of agricultural crops. The interest in the antiphytoviral activity of HYs and EOs of species of the genus Veronica is a continuation of research on the biological activities of species of this genus. Our hypothesis of antiphytoviral activity of FVCs of Veronica species is supported by the fact that volatile components of aromatic plant species can stimulate plant defense responses against infections by various pathogens, including viruses [21,22]. We found that most published data refer to the effects of EOs on animal viruses rather than plant viruses [23,24]. Valuable effects of EOs have been documented, such as antiproliferative, antimicrobial, antioxidant, insecticidal, and other effects [25][26][27], and new findings, such as antiphytoviral activity, have been recently documented [21,28,29].
EOs and their active natural compounds have antibacterial properties against foodborne bacteria and their applications in food preservation could provide alternatives to conventional bactericides and fungicides. Several research studies discovered that shelf-life of fruits and vegetables can be substantially extended when applying specific essential oils [30]. Consequently, the antibacterial effectiveness of five Veronica species was assessed against emerging opportunistic pathogens, among which were clinically isolated multidrug-resistant ESKAPE strains as well as those commonly associated with skin and wound infections and food poisoning outbreaks. Notably, the most recent data on the evidence of the global burden of antimicrobial resistance show that multidrug-resistant bacterial infections are among the major causes of death for people of all ages, resulting in a total of 4.95 million deaths in 2019 [31]. Thus, clinical strains of high-priority pathogen methicillin-resistant Staphylococcus aureus (MRSA) [32] as well as the most common foodborne pathogens Listeria monocytogenes and Bacillus cereus [33] were included in this study.
The aim of this study was to establish a comparative chemical characterization and biological activity potential of FVCs from five Veronica species. To achieve this goal, three objectives were defined: (1) comparison of the volatile profiles of the total extraction products contained in the EO and HY fractions, (2) screening of antiphytoviral activity against TMV, and (3) antibacterial activity against common foodborne pathogens.

Plant Material and Extracts-Review Protocol
Free volatile compounds (FVCs) were extracted from five Veronica species listed in Table 1 (herbarium specimens are presented in the Supplemental Material, Figures S1-S5), and information on their composition has been published in previous articles [13][14][15]. Extracts were obtained by hydrodistillation in a Clevenger apparatus (HD) (Šurlan, Medulin, Croatia) and by microwave-assisted extraction (MAE) (Milestone 'ETHOS X' microwave laboratory oven, 1900 W maximum, Sorisole, Italy). The distillate consists of two layers: essential oil (EO) collected in a side tube using a pentane/diethyl ether trap and a water layer (hydrosol). Identification of FVCs was performed by gas chromatography-mass spectrometry (model 3900; 2100 T; Varian Inc. Lake Forest, CA, USA). Method for FVC analyses is presented in detail in a paper by Dunkić et al. [14].

Antiphytoviral Activity Assay
The above-mentioned extracts were tested on local host plants infected with tobacco mosaic virus (TMV). The species D. stramonium L. was used as a local host for TMV because it develops clearly visible lesions 3-4 days after inoculation. Nicotiana tabacum L. cv. Samsun, which was systemically infected with TMV, was used for virus propagation and virus inoculum production. The experiments were conducted when the local hosts reached the 5-6 leaf stage. Care was taken to ensure that the experimental plants were as uniform in size as possible. The inoculum prepared from systemically infected leaves was diluted with phosphate buffer to obtain 10 to 40 lesions per inoculated leaf. The concentrations (mg/mL) of FVCs from the EO and HY fractions of Veronica species are listed in Table 2 (stock solutions). The HY was applied as an undiluted spray solution to the leaves of D. stramonium plants for two consecutive days. Leaves were dusted with silicon carbide (Sigma-Aldrich, St. Louis, MO, USA) and inoculated with a prepared inoculum. Antiphytoviral activity of HYs was evaluated by the percentage inhibition of the number of local lesions on the leaves of the treated and control plants. The stock solutions of EOs were added to the virus inoculum (500 ppm) and simultaneously inoculated to the leaf halves of the experimental plants. The control leaf halves were inoculated with the same concentration of virus inoculum. The percentage of inhibition was calculated by comparing the number of lesions on the EO-treated and control leaf halves [21].  [34,35]. Strains were tested for antibiotic susceptibility using Etest (AB Biodisk, Sweden) and the VITEK 2 system (bioMérieux, France). Bacterial cells were stored at −80 • C and subcultured on tryptic soy agar (Biolife, Milano, Italy) before testing.

Broth Microdilution Testing
Antibacterial activity was tested using the broth microdilution assay following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for bacteria [36].
EO fractions were diluted in 5% dimethyl sulfoxide (DMSO). Two-fold dilutions of EO-and HY-derived FVC were tested at the maximal concentrations listed in Table 2. Assays were performed in 96-well microtiter plates as previously described [34,35]. In brief, exponentially grown bacterial cultures in Mueller-Hinton broth (Biolife, Milano, Italy) were spectrophotometrically adjusted to a density of 10 6 CFU/mL, added to serial twofold dilutions of FVC in a final volume of 100 µL per well, and incubated at 37 • C for 18 h. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration showing no visually detectable bacterial growth (e.g., turbidity in the wells) and was the consensus value of the experiment performed in triplicate. To determine the minimum bactericidal concentration (MBC), aliquots from the wells corresponding to the MIC, 2× MIC and 4× MIC were plated on Mueller-Hinton agar (Biolife, Milano, Italy) plates. After incubation at 37 • C for 18 h, MBC was determined as the lowest concentration resulting in 99.9% killing of the initial inoculum.
Data on the susceptibility of the microbial strains used in this study were published previously [37,38].

Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 9. All data were expressed as mean ± SD (standard deviation); n = 3. statistical significance was determined by t-test. Differences were considered significant at * p < 0.05.

Free Volatile Compounds of Five Veronica Species
Free volatile compounds (FVC) were extracted from five Veronica species (family Plantaginaceae): V. arvensis L., V. chamaedrys L., V. montana L., V. serpyllifolia L., and V. persica (Table 1, Figures S1-S5) as explained in the Section 2. A total of 20 samples were analyzed, which means that four samples were made for each of the five Veronica spp. studied, and all extracts were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Complete information on the composition of EOs and HYs for all five Veronica species studied can be found in previous articles [13][14][15]. Tables 3 and 4 show the main constituents with the largest relative proportions for all selected species. The major compounds extracted in EOs with HD/MAE are listed in Table 3, and the compounds extracted in HYs with HD/MAE are listed in Table 4. The compounds (E)-caryophyllene, caryophyllene oxide, hexadecanoic acid, hexahydrofarnesyl acetone, and phytol are the most abundant components in EO. In the composition of HY, the common components of all Veronica species studied are caryophyllene oxide, benzene acetaldehyde, and (E)-β-damascenone. In Tables 3 and 4, the main constituents of the extract composition are highlighted because FVCs, as secondary metabolites, have a wide chemical diversity and are represented differently in each taxa, so it is important to highlight them [39]. In this study, we tested the antiphytoviral and antibacterial activity of the total extracts EO and HY from five Veronica species. When extracts that are a mixture of compounds have a greater biological effect than the sum of the individual effects of the compounds, a synergistic effect occurs [40]. In this study, we investigated the biological potential of the natural bioactive components that make up the mixture, i.e., their synergistic effect. Table 3. Main constituents (%) of essential oils (EOs) of five Veronica species studied. HD, Clevenger hydrodistillation; MAE, microwave-assisted extraction [14].

Antiphytoviral Activity of Veronica Species
The antiphytoviral activity of the extracts containing volatiles of Veronica species confirmed our hypothesis, as the results showed a significant reduction of viral infection on the leaves of local host plants. Local lesions on leaf halves inoculated simultaneously with EO and virus were compared with control leaf halves. Results showed that among the EOs, V. chamaedrys EO obtained by MAE was the most effective in reducing viral infection, with an inhibition rate of 68.89% (Figure 1a). Aside from quantitative and qualitative composition that reflects activity, HYs and EOs also have a different mechanism of action, as already reported in the literature. In the study on the antiviral activity of EOs from 29 autochthonous Chinese aromatic plant species, inhibitory activity on TMV infection was described for 13 species, with inhibition rates ranging from 21.0% to 77.9% [42]. Authors described that the EOs of all species showed weaker protective effect than inactivating effect. EOs extracted from Melaleuca alternifolia and Plectranthus tenuiflorus species and applied as spray solution to Nicotiana glutinosa plants before inoculation also showed antiviral activity against TMV [29,43]. Several studies have found that plants are able to initiate a defense response to viral infection using a wide range of regulatory mechanisms and the phenylpropanoid pathway plays a role in such defense strategies [44,45]. Phenylalanine ammonia lyase (PAL) is involved in the initial steps of this metabolic pathway, and the associated gene is known to be involved in plant responses to biotic stresses via transcriptional regulation, leading to the synthesis of several specialized metabolites that play a role in protecting plants from pathogens [46,47]. Taglienti at al. [44] described a significant upregulation of PAL transcription level during treatment when EO or HY of Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis were applied to Cucurbita pepo plants simultaneously or after inoculation of tomato leaf curl New Delhi virus. An induction of PAL gene expression was recorded 12 days post inoculation and then was restored to the level of the untreated control, indicating an early defense response of the plant to the virus infection that may have been enhanced by the treatments.
Based on the above literature findings and supported by the results of our research group, we present that EOs and HYs of Veronica species can reduce viral infection of plants. We assume that EOs, when inoculated simultaneously with the virus, are likely to cause direct inactivation of virus particles and thus are likely to interrupt an early event in the virus replication cycle, as opposed to stimulating the host defense response as hypothesized for the activity of HYs during pretreatment. Our focus on finding volatile compounds with beneficial effects aims to find EOs and HYs that can be used as natural phytotherapeutics. This approach leads us to an ecologically desirable application of all components of the distillation process and, furthermore, to a practical use of extracts that are completely natural and harmless to the environment. As previously reported, different extraction methods affect the composition of volatile components of EOs and HYs [14,48,49] and our results show for the first time that the extraction method also affects the biological activity of the extract. Therefore, the extraction method should be targeted to achieve maximum potential of biological activity of the natural extract. In this study, we Regardless of the higher concentration of V. chamaedrys EO obtained by HD compared to MAE (Table 2), the EO of this species was significantly less effective when extracted by HD, suggesting that the choice of extraction method should be targeted in the context of further use of the natural extract. This activity could be explained with the qualitative as well as quantitative composition of extracts obtained by different methods. Differences in the composition of volatiles obtained by different extraction methods have already been described [11,41]. The compounds (E)-caryophyllene, caryophyllene oxide, hexadecanoic acid, hexahydrofarnesyl acetone, and phytol (Table 3) are the same in composition of the EOs of all Veronica species studied, obtained by both extraction methods. However, the percentages of these components differ significantly depending on the extraction method. For example, in the species V. arvensis, phytol was identified with threefold higher percentage with MAE (22.57%) than with HD (7.54%). On the other hand, in the species V. serpyllifolia twice as much phytol was identified with the extraction method HD (39.79%) compared to MAE, where 18.72% was identified (Table 3). In the composition of HY, the common components of all Veronica species studied are caryophyllene oxide, benzene acetaldehyde, and (E)-β-damascenone (Table 4). Again, we did not observe any regularities in the percentages of identification of these components depending on the extraction method. The most similar percentage of identification in the composition of HY in the species V. chamaedrys is for the compound α-muurolol (23.16% in HD and 22.45% in MAE) ( Table 4) [13][14][15]. The described differences in volatile composition are reflected in the antiphytoviral activity. The present results suggest that MAE is the preferred extraction method for antiphytoviral use of V. chamaedrys EO. Out of the other Veronica species tested, the EO of V. arvensis extracted by both methods was effective in reducing viral infection, with inhibition rates of 57.89% with MAE and 53.97% with HD, respectively, but there was no statistical difference between the activity of the two extracts, suggesting that both methods are useful in terms of the antiphytoviral activity of the EO of this species. The EOs of the remaining three species tested were more effective in inhibiting TMV infection when extracted by the HD method. Among these oils, the oil of V. persica showed an inhibition rate of 58.67% on HD (Figure 1a) and was significantly more effective than the same oil extracted using MAE. A similar difference between the activity of the EOs extracted by the two methods was observed for the oil of V. montana, which showed an inhibition rate of 42.00% and 18.49% on HD and MAE, respectively. The EO of V. serpyllifolia showed no such difference between the activity of the oils extracted by MAE and HD.
The antiphytoviral activity was confirmed and was even more pronounced when the HYs of the tested Veronica species were applied as a spray solution to the local host plants two days before virus inoculation (Figure 1b). HYs as aqueous solutions are harmless to the plants and were applied without dilution after both distillation processes. Comparison of antiviral activity of the HYs obtained by MAE and HD showed that the latter method yielded more effective hydrosols, as promising antiviral activities of 79.21% (V. arvensis and V. chamaedrys) and 71.13% (V. persica) were observed. From the data obtained, it can be concluded that HD is the preferred extraction method when HYs of these three Veronica species are used as possible antiviral agents. MAE and HD showed no significant difference in the antiviral activity of the extract when the hydrosols of V. montana and V. serpyllifolia were tested, as promising activities ranging from 64.42% to 76.38% were observed.
Aside from quantitative and qualitative composition that reflects activity, HYs and EOs also have a different mechanism of action, as already reported in the literature. In the study on the antiviral activity of EOs from 29 autochthonous Chinese aromatic plant species, inhibitory activity on TMV infection was described for 13 species, with inhibition rates ranging from 21.0% to 77.9% [42]. Authors described that the EOs of all species showed weaker protective effect than inactivating effect. EOs extracted from Melaleuca alternifolia and Plectranthus tenuiflorus species and applied as spray solution to Nicotiana glutinosa plants before inoculation also showed antiviral activity against TMV [29,43]. Several studies have found that plants are able to initiate a defense response to viral infection using a wide range of regulatory mechanisms and the phenylpropanoid pathway plays a role in such defense strategies [44,45]. Phenylalanine ammonia lyase (PAL) is involved in the initial steps of this metabolic pathway, and the associated gene is known to be involved in plant responses to biotic stresses via transcriptional regulation, leading to the synthesis of several specialized metabolites that play a role in protecting plants from pathogens [46,47]. Taglienti at al. [44] described a significant upregulation of PAL transcription level during treatment when EO or HY of Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis were applied to Cucurbita pepo plants simultaneously or after inoculation of tomato leaf curl New Delhi virus. An induction of PAL gene expression was recorded 12 days post inoculation and then was restored to the level of the untreated control, indicating an early defense response of the plant to the virus infection that may have been enhanced by the treatments.
Based on the above literature findings and supported by the results of our research group, we present that EOs and HYs of Veronica species can reduce viral infection of plants. We assume that EOs, when inoculated simultaneously with the virus, are likely to cause direct inactivation of virus particles and thus are likely to interrupt an early event in the virus replication cycle, as opposed to stimulating the host defense response as hypothesized for the activity of HYs during pretreatment. Our focus on finding volatile compounds with beneficial effects aims to find EOs and HYs that can be used as natural phytotherapeutics. This approach leads us to an ecologically desirable application of all components of the distillation process and, furthermore, to a practical use of extracts that are completely natural and harmless to the environment. As previously reported, different extraction methods affect the composition of volatile components of EOs and HYs [14,48,49] and our results show for the first time that the extraction method also affects the biological activity of the extract. Therefore, the extraction method should be targeted to achieve maximum potential of biological activity of the natural extract. In this study, we demonstrated the possibility of developing natural resources from the genus Veronica for crop protection. Based on that mentioned above, we highlight five species of the genus Veronica with their volatiles as a valuable source of natural products with beneficial effects and point out their antiphytoviral activity here for the first time.

Antibacterial Activity of Five Veronica Species
The antimicrobial potential of various plant extracts from a considerable number of species from the genus Veronica was previously explored and summarized [7,8]. In this study, we aimed to evaluate the antibacterial activity of FVCs from EO and HY fractions obtained by two extraction methods from five Veronica species using a broth microdilution assay against a wide range of human opportunistic pathogens associated with skin and wound infections, as well as foodborne pathogens. Bacterial growth was not affected by HY fractions, regardless of the Veronica species. However, it should be noted that the maximum concentrations of the fractions tested in this study were low and ranged between 1.83 and 2.91 mg/mL, respectively. Therefore, these results agree with Dunkić et al. who found that aqueous extracts of Veronica spicata (at a concentration of 10 mg/mL) were ineffective against several bacterial and fungal pathogens [50]. In addition, Stojković et al. reported Veronica montana water extract to be active at ≥7.5 mg/mL [51].
On the other hand, the EO-derived FVCs exhibited an antibacterial effect which varied depending on the Veronica species and mostly showed similar activities depending on the isolation method used (Table 5). In general, EOs were uniformly more effective against Gram-positive bacteria compared to Gram-negative ones, which in most cases were not successfully inhibited at the maximum concentrations tested. This was expected, as several authors reported elevated MIC values of various extracts of Veronica species against Gramnegative bacteria [7]. Notably, Dunkić et al. reported better activity against Gram-positive species for methanolic and ethyl acetate extracts of Veronica spicata, and Hassan and Ullah for ethyl acetate extracts of V. biloba [50,52]. Similarly, Živković et al. documented that Gram-positive bacteria, especially Staphylococcus aureus, were most sensitive to methanolic extracts of Veronica urticifolia [53]. In addition, ethanolic extracts of Veronica officinalis, V. teucrium, and V. orchidea showed stronger antibacterial activity against Gram-positive bacterial pathogens, including several Listeria spp. and Bacillus cereus [10]. This pattern of susceptibility has been associated with the different structure of the cell wall in Gram-positive bacteria and the presence of a complex outer membrane in Gram-negative bacteria, which reduces the uptake of compounds into the cells while at the same time supporting their multifactorial efflux pump system [52,54,55]. Nevertheless, it is important to note that different extraction methods were used in the aforementioned studies, and this paper represents the first report of the antibacterial activity of FVCs from EO fractions obtained by HD and MAE. Notably, among the tested EO fractions, those from V. chamaedrys, V. serpyllifolia, and V. arvensis exhibited the strongest antibacterial effect (Table 5). To the best of our knowledge, an overview of the scientific literature did not reveal any available data on the antibacterial activity of EO-associated FVCs from V. chamaedrys, although this species is a long-known traditional medicinal plant [56], whose aqueous and alcoholic extracts were shown to have anti-inflammatory and antioxidant effects [8]. More importantly, nothing has been found in the literature about any type of V. arvensis fractions investigated in the context of biological activity, including antibacterial. As for V. serpyllifolia, only one study reported the radical scavenging activity of its water extract [57].
In this study, Listeria monocytogenes was the most sensitive bacterium to EO fractions from the five Veronica species (Table 5). This emerging foodborne pathogen, which has been associated with outbreaks worldwide and mortality rates as high as 20% [58], was successfully inhibited at concentrations ranging from 0.32 to 6.13 mg/mL, with V. chamaedrys, V. serpyllifolia, and V. arvensis active at concentrations below 1 mg/mL. On the other hand, S. aureus was the least sensitive among the Gram-positive bacteria with MIC values ranging from 4.89 to 42.89 mg/mL. However, it is noteworthy that the clinical MRSA strain was inhibited at the same MIC values as the antibiotic-sensitive ATCC strain in most cases. Notably, S. aureus is one of the most common hospital and community-acquired opportunistic pathogens worldwide [59]. It frequently causes skin, soft tissue, and bloodstream infections; 70% of which are due to MRSA strains that are resistant to most β-lactam antibiotics as well as those from other classes [60].
Moreover, the results of the susceptibility testing did not provide conclusive evidence of uniformly enhanced antibacterial activity when a particular isolation method was used. Although the stock solutions of the fractions differed depending on the isolation method employed (Table 2), the fractions of V. serpyllifolia exhibited very similar MIC and MBC values (Table 5), successfully killing S. pyogenes, S. agalactiae, E. faecalis, and L. monocytogenes within a range of 0.85 to 1.77 mg/mL. In the case of V. chamaedrys, the fraction derived by MAE showed considerably better activity only against L. monocytogenes and E. faecalis at MIC values of 0.32 mg/mL and 1.28 mg/mL, respectively. On the other hand, the EO fractions of V. arvensis exhibited somewhat better activity when the HD method was used, except in the case of L. monocytogenes, whose growth was inhibited at 0.53 mg/mL. Nevertheless, the overall range of observed MIC values was within 2-fold for the majority of bacteria tested.
Previously, studies investigating Veronica extracts showed that the presence of various biologically active compounds is species-specific and dose-dependent, but those exhibiting antibacterial effect mainly include glycosides, flavonoids, iridoids, and saponins [7,9]. Moreover, it was hypothesized that phenolic compounds in particular may contribute to antibacterial activity by producing a synergistic effect with other bioactive compounds [10,50,51]. In Tables 3 and 4 the main constituents of EOs and HYs, previously analyzed and published, are reviewed [14]. These compounds were found to exert an antibacterial effect. In fact, phytol exhibits various biological activities, including antimicrobial, cytotoxic, antioxidant, autophagy-and apoptosis-inducing, anti-inflammatory, and immunomodulatory [61]. Compound (E)-caryophyllene is known for its anti-inflammatory and anesthetic properties, while Montanari et al. also pointed out that a high content of sesquiterpenes (up to 35%) in EOs from Verbenaceae species corresponds with their moderate antibacterial activity [62].
Although the exact mechanism of action of terpenes has not yet been elucidated, there is increasing evidence that these compounds, including eugenol [63], act rapidly against bacterial cells via highly reactive hydroxyl groups that form hydrogen bonds with the active sites of target enzymes, leading to their inactivation [64,65], as well as acting via changes in membrane ion channels (Na + , K + , Ca 2+ , or Cl − ) that cause cell rupture and a leakage of the cellular contents [66]. Considering the chemical composition of the EO fractions [14], we speculate that the high amounts of oxygenated sesquiterpenes (13.2 to 40.44%), especially caryophyllene oxide and eudesmol (14.11% and 19.98% in the HD-EO fraction of V. arvensis, respectively), play an important role in their antimicrobial activity (Table 5). Moreover, these fractions were rich in diterpene phytol, of which the HD-EO fractions of V. serpyllifolia and V. chamaedrys accounted for 39.79% and 31.66%, respectively. It is also important to note that the most active EO fractions uniformly contained >2% phenolic compounds, of which the HD fraction from V. arvensis contained a total of 6.99% with 2.16% eugenol [14]. However, as we have already noted, there is no clear pattern of enhancement of their antibacterial activity with respect to the isolation method used, as the choice of method does not uniformly enrich or reduce the amount of a particular biologically active volatile compound in EO fractions [14].

Conclusions
The Plantaginaceae family is one of the most important families with biopesticidal activity. In our previous study, the free volatile compounds (FVCs) of five Veronica species contained in essential oil (EO) and hydrosol (HY) were obtained by classical (Clevenger apparatus, HD) and green hydrodistillation (microwave-assisted extraction, MAE) and the compositions of the extracts were compared. Continuing on this research, in the present study we investigated the antiphytoviral and antibacterial activities of FVCs. The results showed that among the EOs, EO-MAE extracted from V. chamaedrys was the most effective in reducing viral infection. More effective antiphytoviral activity was shown by the extracts of HY-HD, especially in V. arvensis, V. chamaedrys, and V. persica. In general, for the majority of tested extracts, hydrosols showed better antiphytoviral activity than EOs. Extracts from the five Veronica species were also tested by broth microdilution against a wide range of human opportunistic pathogens associated with skin and wound infections and foodborne pathogens. HY fractions had no effect on bacterial growth, regardless of the Veronica species tested, likely due to the fact that the maximum concentrations of the HY fractions tested in this study were low. EOs showed significant antibacterial activity independent of the extraction method. Only V. chamaedrys, the EO-MAE fraction, showed significantly better activity against Listeria monocytogenes and Enterococcus faecalis than EO-HD. The EO-HD fraction of V. arvensis also showed slightly better antibacterial activity. We can conclude that by combining extracts and using different extraction methods, valuable bioproducts can be obtained for safe use in agricultural production, which have great benefits for human health and the environment as a whole. The results of this study indicate the potential use of all parts of the extracts of the studied Veronica species as natural phytotherapeutics relevant to biological control of pests, with significant ecological benefits.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/agriculture13081517/s1, Figure S1: Herbarium specimen of the species Veronica arvensis L.; Figure S2: Herbarium specimen of the species Veronica chamaedrys L.; Figure S3: Herbarium specimen of the species Veronica montana L.; Figure S4: Herbarium specimen of the species Veronica serpyllifolia L.; Figure S5: Herbarium specimen of the species Veronica persica Poir.

Conflicts of Interest:
The authors declare no conflict of interest.