Antiviral Activity of Selected Essential Oils against Cucumber Mosaic Virus

The aim of the study was to assess the antiviral activity of selected essential oils (EOs) against Cucumber mosaic virus (CMV), both in vitro and in vivo. The observations were made using Chenopodium quinoa as a local host. The EOs were obtained from Greek oregano, thyme, and costmary. Their chemical composition was determined using GC/FID followed by GC/MS. The dominant compound in oregano EO was carvacrol (59.41%), in thyme EO—thymol (59.34%), and in costmary EO—β-thujone (90.60%). Among the analysed EOs, thyme EO exhibited the most promising effects against CMV. However, its activity was influenced by the time of application. In an in vivo experiment, thyme EO showed protective (pre-inoculation) rather than curative (post-inoculation) activity.


Introduction
Viral plant diseases can be found worldwide. Plant viruses infect many crops, causing serious losses in their production. Protection of crops against viruses is difficult. In general, two methods of crop protection against viruses are employed in plant cultivation. The first one, as applied especially in the case of vegetatively propagated plants, consists of ensuring that a sanitarily certified propagation material is used, the second one being chemical control of insect vectors. Another method involves the use of resistant varieties developed by breeding. However, effective crop protection with this method is possible only for a limited number of viruses. Additionally, it is possible to eliminate viruses in vitro, which is accomplished with different techniques (e.g., thermotherapy or meristem culture) which are all expensive, time-consuming, and are not suitable for all plant/virus pathosystems [1,2]. Thus, the search for alternative methods of crop protection against plant viruses is of great significance.
Essential oils (EOs), due to their wide range of biological activities, have been studied extensively. Since the Middle Ages, they have seen widespread use in bactericidal, virucidal, fungicidal, antiparasitical, and medicinal applications. At present, they are especially common in pharmaceutical, sanitary, cosmetic, agricultural, and food industries. Moreover, EOs are of great importance in several fields, including plant protection against diseases [3][4][5][6][7]. Although limited, current knowledge about the antiviral effects of EOs indicates their potential to control the spread of viral infections [6,8]. A number of recent reports have provided data on the activity of EOs against plant viruses [4,[9][10][11][12][13][14][15][16][17][18][19]. However, this field of study is still insufficiently explored and further research is required to enable a more complete understanding of the mechanisms behind the antiviral activities of EOs [1].
The detailed mechanism of such activity is yet to be fully explored. It has been hypothesized that EO components could either directly inactivate viral particles or induce resistance/tolerance response in the host [2]. The mechanisms of action of EOs are versatile

Evaluation of the Antiviral Activity of the EOs against the CMV-S21 Isolate (In Vitro)
Greek oregano, thyme, and costmary EOs were applied on the Ch. quinoa host plants simultaneously with the application of the virus, which reduced the number of local lesions on the CMV-infected plants. Significant differences were observed in the activity of thyme EO at the concentrations of 2000 ppm, 3000 ppm, 4000 ppm, 5000 ppm, and 6000 ppm against the CMV-S21 isolate compared with the effect of Greek oregano and costmary EOs. Thyme EO was the most effective in reducing the number of local lesions on the Ch. quinoa leaves and showed inhibition of local lesions at the level 30.1-79.5%. The strongest activity of thyme EO was recorded at the concentration of 6000 ppm, and the mean number of spots on four inoculated leaves was 30.4. (inhibition of local lesions was 79.5%). No significant differences were observed between the activity of thyme EO (1000 ppm), Greek oregano EO (3000-6000 ppm), or costmary EO (6000 ppm) against the CMV-S21 isolate. The number of local spots visible on plant leaves after inoculation with the CMV-S21 isolate (control group 3) was not significantly different from the number of spots on the Ch. quinoa leaves after inoculation of plants with the CMV-S21 isolate with the addition of costmary EO (500-5000 ppm), Greek oregano EO (500-3000 ppm), or thyme EO (500 ppm) ( Table 2). The average absorbance (OD 405 nm ) of samples from the experimental group was lower than that of the samples from control group 3. The average absorbance (OD 405 nm ) of all the EOs tested against CMV-S21 isolate increased with the increase in their concentrations. The average absorbance (OD 405 nm ) for samples of plants infected with the CMV-S21 isolate with the addition of thyme EO (500-6000 ppm) was lower compared with the average absorbance for Greek oregano and costmary EOs. The average absorbance (OD 405 nm ) was the lowest for samples of plants infected with the CMV-S21 isolate with the addition of thyme EO at the concentration of 6000 ppm (Table 3).

Evaluation of the Antiviral Activity of the EOs against the CMV-S21 Isolate (In Vivo)
Thyme EO showing the greatest percent of inhibition of local symptoms on the Ch. quinoa plants in the in vitro experiment were chosen for in vivo testing.
The samples of Ch. quinoa plants from Test A and Test B, control groups I and II were analysed by DAS-ELISA test. The DAS-ELISA test results are presented in Table 4.

Spot number
Thyme essential oil concentration (ppm) The samples of Ch. quinoa plants from Test A and Test B, control groups I and II were analysed by DAS-ELISA test. The DAS-ELISA test results are presented in Table 4. The average absorbance (OD 405 nm ) of samples from the experimental group was lower than that of the samples from control group II. The average absorbance (λ = 405 nm) readings for the samples from the plants treated with thyme oil 24, 48, and 72 h before the inoculation with CMV were lower compared with those for the samples from the plants on which the EO was applied 24, 48, and 78 h after the inoculation with CMV ( Table 4).
The application of thyme EO (6000 ppm) on Ch. quinoa leaves before the inoculation with CMV proved more effective than the application of the EO after the inoculation with CMV. These differences were statistically significant. No significant differences were observed in the number of local lesions visible on the leaves of Ch. quinoa plants treated with thyme EO 24, 48, and 72 h before the inoculation with CMV. The number of local lesions was significantly lower when application of thyme EO was performed 72 h after the inoculation with CMV compared with the application of the EO after 24 h or 48 h. The effect of thyme EO applied on the leaves 24 h and 48 h after the inoculation was not statistically different ( Figure 2).

Discussion
Chemically, essential oils are multi-component mixtures of monoterpenes, sesquiterpenes, and their derivatives, including aromatic derivatives. These substances can be in the form of alcohols, ketones, aldehydes, esters, or ethers. Usually, a single essential oil contains several dozens of compounds with different concentrations and activities. Large variation in the chemical composition of EOs determines a versatile range of their applications. Chemical polymorphism among aromatic plants is a widely described phenomenon [31] The number of individual compounds and their proportion in a particular EO is variable and depends on many factors, including genetic variations of the species, the age of the plant, the type of raw material obtained, geographical location of cultivation, the time (season) and conditions of harvest, the environmental conditions of growth, post-harvest treatment of raw materials, and the method of isolation. In the vast majority of EOs, however, it is possible to distinguish one to three dominant compounds responsible for a specific aroma and biological activity of EOs [21,24,[32][33][34][35][36].
The aim of the first experiment in the present study was to verify whether Greek oregano, thyme, and costmary EOs exhibit antiphytoviral activity against CMV.
A common feature of Greek oregano and thyme EOs is a high content of monoterpenes, including phenolic monoterpenes, oxygenated monoterpenes, and monoterpene hydrocarbons. However, there were significant differences in the composition of the two EOs. Greek oregano EO had a higher proportion of carvacrol (59.41%) and γ-terpinene (19.73%), while the content of thymol amounted to 1.19%. Conversely, in thyme EO, thymol (59.34%) was the dominant compound, with the content of carvacrol and γ-terpinene amounting to 2.41% and 12.78%, respectively. Moreover, Greek oregano EO contained a higher percentage of oxygenated monoterpenes, whereas thyme EO contained a higher percentage of sesquiterpenes. In costmary EO, the dominant compound was β-thujone (90.60%), classified as an oxygenated monoterpene. The content of monoterpene hydrocarbons, phenolic monoterpenes, and sesquiterpenes was low in this EO compared with the content in Greek oregano and thyme EOs.
Apart from the dominant compounds, the Greek oregano EO contained a higher percentage of oxygenated monoterpenes, whereas the thyme EO contained a higher percentage of sesquiterpenes. In the costmary EO, the oxygenated monoterpenes were the dominant component, while the content of monoterpene hydrocarbons, phenolic monoterpenes, and sesquiterpenes was low compared with the content of these compounds in the Greek oregano and thyme EOs. Both phenolic monoterpenes and monoterpene hydrocarbons were present in the Greek and thyme EOs in high percentages, while the costmary EO had a high content of oxygenated monoterpenes, which indicates a relationship between the class of chemical compounds in which it was present in a particular EO in the largest proportion and how effective was the antiviral activity it exhibited. In addition to the main component of the tested EO obtained from a specific plant species, its biological properties and, thus, its antiviral activity, will also be affected by the percentage content of the individual components [28].

Despite a wide spectrum of biological activities of EOs, only a limited amount of information is available about their effect on CMV.
A number of similarities was observed between the composition and activity of EOs investigated in this study and the currently available literature data. According to Bezić et al. [4], the application of Satureja montana EO on Ch. quinoa and Ch. amaranticolor host plants simultaneously with the inoculation with CMV reduced the number of local lesions by 24.1%. The main components of S. montana EO were thymol and carvacrol. Thymol was more effective in reducing the percentage of CMV infection (reduction by 33.2%), while the percentage reduction of CMV infection by carvacrol amounted to 28.3% [4]. Moreover, the EOs of several Teucrium species (T. polium, T. flavum, T. montanum, T. chamaedrys, and T. arduini), rich in monoterpenes and sesquiterpenes, significantly reduced the percentage of CMV infection of Ch. quinoa plants (reduction by 22.9-43.4%) [4,24]. Sesquiterpenes and monoterpenes were also present in relatively high percentages in Micromeria fructiculosa and M. graeca EOs, thus reducing the number of local lesions on Ch. quinoa inoculated with CMV by 23.8-43.6% [26,28,29]. Additionally, EOs of both Eryngium alpinum and E. amethystinum significantly reduced the number of local lesions on Ch. quinoa inoculated with CMV (reduction by 77.8% and 80.5%, respectively). The EOs of both species contained high percentages of oxygenated sesquiterpene compounds [23]. The experiments revealed that the effectiveness of thyme essential oil against CMV depends on its concentration. Local lesion inhibition of 50% was achieved by applying the thyme EO at a concentration of 3000 ppm, whilst the inhibition of 80% was achieved by using a concentration of 6000 ppm.
The results obtained in this study indicate that the time of application of thyme EO has a significant influence on its activity against the CMV infection. The best inhibitory effect was obtained through protective (prior to virus inoculation) rather than curative (postinoculation) treatment. According to Helal [19], the maximum protection and inhibition percentage (91.5%) was observed 24 h before the application of the thyme essential oil (3000 ppm) on cucumber plants. These results correspond with those of Vuko et al. [29] who treated the Ch. quinoa plants with Micromeria croatica essential oil 24, 48, and 72 h prior to the inoculation with CMV. Although all pre-treatments significantly reduced the number of local lesions, the strongest antiviral effect was manifested after the treatment that was performed 72 h prior to the inoculation. The percentage reduction of the number of local lesions ranged between 66.8% and 71.4%. According to Dunkić et al. [23], preinoculation treatment with both the Eryngium alpinum and E. amethystinum EOs significantly reduced the number of local lesions, with the percentage reduction amounting to 77.8 and 80.5%, respectively. The antiviral effect of EOs appears to involve direct inhibition of virus replication, or indirect inhibition through induction of systemic resistance of the host plant against the virus, which may persist for long periods depending on both plant species and the virus strain [19].
All of the studied EO components, some of which are present as major constituents and some of which are present in relatively small amounts, can have a synergetic effect and may contribute to the antiviral efficacy of the EOs. Antiviral testing of many EOs could help us gain insight into the relationship between the EO composition and their antiviral efficiency. The results obtained can be a starting point for further research into the antiphytoviral activity of essential oils and individual components of the oils. Understanding the mode of such activity may help find and adjust these natural substances for possible use in the control of viral plant diseases [4].
To our knowledge, this is the first study to address this topic in Poland.

Plant Raw Materials Used for Distillation of EOs
The EOs used in the study were obtained from herbs of three species

EOs Extraction and GC-MS/GC-FID Analysis
Essential oils were extracted according to European Pharmacopoeia, with modifications [37]. Fifty grams of air-dried raw material was used for hydrodistillation for 3 h using a Deryng-type apparatus. Until the analysis, the samples were stored in dark vials at 4 • C.
Analysis of essential oils were carried out by gas chromatography (GC) coupled with mass spectrometry (MS) and flame ionization detector (FID). The qualitative and quantitative analysis was carried out by means of an Agilent Technologies 7890A gas chromatograph equipped with FID and MS Agilent Technologies 5975C Inert XL_MSD with Triple Axis Detector (Agilent Technologies, Wilmington, DE, USA). Details of the operation conditions were given previously by Bączek et al. [38]. Capillary, polar column HP 20M (25 m × 0.32 mm × 0.30 µm) (Agilent Technologies, Wilmington, DE, USA) was applied. Separation conditions were as follows: oven temperature isotherm at 60 • C for 2 min, temperature rising at a rate of 4 • C per min, from 60 • C to 220 • C, then held isothermal at 220 • C for 5 min. The carrier gas (He) flow was 1.1 mL/min. The split ratio was 1:50. Diluted samples (1/100 v/v, in n-hexane:isopropanol) of 1 µL were injected at 210 • C by auto sampler. Ion source temperature was −220 • C, ionization voltage was 70 eV, and the range of mass spectra scanning was 40-500 amu. EOs compound identification was based on comparison of mass spectra from the Databases (NIST08, NIST27, NIST147, NIST11, Wiley7N2) and on comparison of retention indices (RI) relative to retention times of a series of n-alkanes (C 7 -C 30 ) (Merck KGaA, Darmstadt, Germany) with those reported in the literature [38]. The percentage share of compounds identified in the EOs was computed by the normalization method from the GC peak areas.
The air-dried herb was hydrodistillated for 3 h using a Deryng-type apparatus. Until the analysis, the samples were stored in dark vials at 4 • C.
Analysis of essential oils was conducted by GC-MS and GC-FID (gas chromatography coupled with mass spectrometry and flame ionization detector).
The qualitative and quantitative analysis was carried out by means of an Agilent Technologies 7890A gas chromatograph equipped with a flame ionization detector (FID) and MS Agilent Technologies 5975C Inert XL_MSD with Triple Axis Detector (Agilent Technologies, Wilmington, DE, USA). Capillary, polar column HP 20M (25 m × 0.32 mm × 0.30 µm) (Agilent Technologies, Wilmington, DE, USA) was applied. Separation conditions were as follows: oven temperature isotherm at 60 • C for 2 min, temperature rising at a rate of 4 • C per min, from 60 • C to 220 • C, then held isothermal at 220 • C for 5 min. The carrier gas (He) flow was 1.1 mL/min. The split ratio was 1:50. Diluted samples (1/100 v/v, in n-hexane:isopropanol) of 1 µL were injected at 210 • C by auto sampler. Ion source temperature was −220 • C, ionization voltage was 70 eV, and the range of mass spectra scanning was 40-500 amu. EOs compound identification was based on comparison of mass spectra from the Databases (NIST08, NIST27, NIST147, NIST11, Wiley7N2) and on comparison of retention indices (RI) relative to retention times of a series of n-hydrocarbons (C 7 -C 30 ) with those reported in the literature. The percentage share of compounds identified in the EOs was computed by the normalization method from the GC peak areas.

Virus and Plant Host
Cucumber mosaic virus (CMV) (Bromoviridae family, genus Cucumovirus), S21 CMV isolate, as well as seeds of the Chenopodium quinoa Willd. host plants were provided by The Department of Virology and Bacteriology, Institute of Plant Protection, National Research Institute, Poznań.
The seeds of Ch. quinoa were sown in trays in a greenhouse at a temperature of 24 • C under a 16 h/8 h light/dark cycle, with watering as required. When the seedlings were large enough to handle, they were potted individually into 15 cm plastic pots containing fresh compost. The host plants were grown in a greenhouse under the same conditions. The experimental plants were selected for inoculation four weeks after sowing when they had eight true leaves. Care was taken to ensure that the experimental plants were as uniform in size as possible.
The viral inoculum was prepared from cucumber leaves infected with the S21 CMV isolate. The plant material was ground with cold inoculation buffer (0.05 M phosphate buffer, pH 7.0) in a cold mortar. The inoculum prepared was used for mechanical inoculation of Ch. quinoa host plant.

Antiviral Effects of the EOs on the Ch. Quinoa Plants
Greek oregano, thyme, and costmary EOs were studied both in vitro and in vivo. In vitro antiviral activity-Equal volumes of solutions containing 500 ppm, 1000 ppm, 2000 ppm, 3000 ppm, 4000 ppm, 5000 ppm, and 6000 ppm of the EOs in Tween 80 and distilled water were added separately to the virus inoculum (20 µg/mL concentration) and immediately used for mechanical inoculation of the Ch. quinoa plants (the experimental group).
Control The protective and curative effect of the evaluated EOs against CMV infection was investigated in vivo according to the method described by Helal

DAS-ELISA Test
DAS-ELISA test [39] was used to estimate the antiviral activity of the EOs against the CMV-21 isolate using a specific antibody from LOEWE Biochemica GmbH (Germany). The plant samples (samples from the experimental group, control groups 1, 2, and 3, Test A and Test B, control groups I and II) were prepared by grinding 0.250 g of fresh plant tissue in an extract buffer in the ratio of 1:10 (w/v) and tested according to the manufacturer's protocol. After 1 h of incubation at room temperature, substrate hydrolysis was measured as a change in absorbance at OD 405 nm using the Infinite ® 200Pro microplate reader (Tecan GmbH, Austria). Samples were considered positive if their optical density (OD 405 nm ) readings were at least twice those of the healthy controls. The average absorbance values of the experimental group and control groups 1, 2, and 3 are presented.

Statistical Analysis
One-way analysis of variance (ANOVA) was performed to investigate the effects of the EOs used at the concentrations of 500, 1000, 2000, 3000, 4000, 5000, and 6000 ppm against the CMV activity. The experiment was conducted in 20 replications. Each replication was one virus-inoculated Ch. quinoa plant treated with one of the 3 tested EOs at the above concentrations, for which the number of local spots present on 4 leaves was determined by counting. The control variant were 20 CMV-inoculated plants. An additional control variant were plants treated with only the 3 tested EOs of different concentrations, and the Student-Newman-Keuls multiple comparison test was used at a significance level of p = 0.05.
To test the relationship between the concentration of an EO and the average number of spots present on 4 leaves of Ch. quinoa after inoculation with the CMV-S21 isolate, regression analysis was performed. The analysis was carried out for the most effective EO (showing the greatest percentage of inhibition of local symptoms on the Ch. quinoa plants).
A linear regression equation was calculated according to the model: where y = average spot number, and x = thyme EO concentration.
One-way analysis of variance (ANOVA) was performed to test the effectiveness of the EO showing the greatest percentage of inhibition of local symptoms on the Ch. quinoa plants in the in vitro experiment depending on the time of its application (24 h, 48 h, or 72 h before inoculation or 24 h, 48 h, or 72 h after inoculation with the isolate S21 CMV. For comparisons of the effects of thyme EO applied on the leaves of the Ch. quinoa plants before and after inoculation, the Student-Newman-Keuls multiple comparison test was used with a significance level of p = 0.05. All statistical analyses were performed using Statgraphics Plus for Windows 4.1.

Conclusions
Among the analyzed EOs, only thyme EO showed significant antiviral activity against CMV. The strongest activity of thyme EO was recorded at the concentration of 6000 ppm and the mean number of spots on 4 inoculated leaves was 30.4. (Inhibition of local lesions 79.5%). However, the activity clearly depended on the method of application and the duration of the application. The application 24 h, 48 h, and 72 h before the virus inoculation gave much better results than the application after the virus inoculation. The presented study is novel and constitutes the first step towards research into future methods of plant protection against viruses.