Antiphytoviral Activity of Sesquiterpene-Rich Essential Oils from Four Croatian Teucrium Species

The purpose of this study was to compare the essential oil profiles of four Croatian Teucrium species (Lamiaceae), as determined by GC and GC/MS, with their antiphytoviral efficiency. A phytochemical analysis showed that T. polium, T. flavum, T. montanum and T. chamaedrys are characterized by similar essential oil compositions. The investigated oils are characterized by a high proportion of the sesquiterpene hydrocarbons β-caryophyllene (7.1–52.0%) and germacrene D (8.7–17.0%). Other important components were β-pinene from T. montanum and α-pinene from T. flavum. The investigated essential oils were proved to reduce lesion number in the local host Chenopodium quinoa Willd. infected with Cucumber Mosaic Virus (CMV), with reductions of 41.4%, 22.9%, 44.3% and 25.7%, respectively.


Introduction
In the flora of Europe the genus Teucrium (Lamiaceae) includes 49 species, of which 13 are widespread in the Croatian flora, including the four species studied in this paper: T. polium L., T. flavum L., T. montanum L. and T. chamaedrys L. [1][2][3][4]. The essential oils of several other species of the genus Teucrium, viz. T. stocksianum ssp. stocksianum [5], T. hyrcanicum [6], T. maghrebinum [7], T. montbtetii ssp. heliotropiifolium [8], T. scoridum [9] and T. salviastrum [4] have been previously investigated. These oils are characterized by the presence of sesquiterpenes such as caryophyllene, caryophyllene oxide, germacrene D, α-humulene, α-muurolene, (E)-β-farnesene and the monoterpene carvacrol. Due to the wide spectrum of biological activities displayed by the essential oils, these compounds are the subject of different researches. The multiple roles of the essential oils and their main components make them natural substances of great importance in several fields such as physiological function of growth, ecological function, development [10], resistance against diseases and insects [11]. They also posses antimicrobial, antiviral, antimycotic, antioxigenic, antiparasitic and insecticidal properties [12][13][14][15][16]. Regarding phytopathogenic viruses, various substances of natural and synthetic origin have been assessed for their antiphytoviral activity [17][18][19][20]. So far, only a few studies have revealed the antiphytoviral activity of pure essential oils [13,17,21,22]. Essential oil of Satureja montana L. ssp. variegata (Host) P.W. Ball as well as its dominant phenol compounds thymol and carvacrol applied simultaneously with the infecting virus, reduced the number of local lesions on both Tobacco Mosaic Virus (TMV) and Cucumber Mosaic Virus (CMV) infected plants [21]. Essential oil of Melaleuca alternifolia (Maiden&Betche) was effective in reducing lesion number in TMV-infected plants [13], while Plectranthus tenuiflorus essential oil inhibited Tobacco Necrosis Virus (TNV) infection [17]. Essential oil from Mediterranean endemic plant Teucrium arduini L. showed inhibitory effect on the development of infections caused by TMV and CMV [22].
The aim of the study was to determine the volatiles of four Croatian grown Teucrium species and evaluate their antiphytoviral effects on the development of local lesions in CMV infected plants.
Cavaleiro et al. [4] reached similar conclusion for endemic species T. salviastrum from Portugal. In the oil of T. polium ssp. capitatum from Crete [8] the most abundant compounds were caryophyllene and the monoterpene carvacrol. Carvacrol was absent in all investigated oils, but in our previous study of T. arduini essential oil carvacol was represented in an amount of 1.6% [22]. The composition of T. polium from Iran [23] was similar to the investigated oils of T. flavum and T. montanum regarding the content of α-and β-pinene. In the present study we also identified limonene as an important compound of essential oils of T. polium (5.9%), T. flavum (7.9%) and T. montanum (4.6%). These monoterpenes were reported as the main constituents in the oil of T. chamaedrys ssp. chamaedrys from Iran [6]. In this study, in relatively high percentages, essential oil from T. flavum contained α-pinene (10.5%), β-pinene (8.4%) and limonene (7.9%), while T. montanum essential oil contained β-pinene (12.3%) and limonene (4.6%).

Antiphytoviral Activity
A comparison (t-test) of the mean number of lesions on the oil-treated Chenopodium quinoa plants with the corresponding control showed that essential oils isolated from the investigated Teucrium species significantly reduced CMV infections. The most effective in reducing local lesion number was oil of T. montanum (44.3%), followed by T. polium (41.4%), T. chamaedrys (25.7%) and T. flavum (22.9%) ( Table 2). The common feature of all investigated oils is the presence of β-caryophyllene and germacrene D in relatively high percentages. Our previous investigation had confirmed that β-caryophyllene is effective in reducing CMV infection [22]. With the exception of T. montanum essential oil, the percentage of β-caryophyllene in the oil correlates with antiviral activity of the oil. Essential oil of T. montanum showed the strongest antiviral activity, although content of β-caryophyllene is lower when compared to the other three oils. The most abundant components from T. montanum essential oil, aside from β-caryophyllene, are germacrene D, β-pinene and limonene and as such they can be suggested to be responsible for the better antiphytoviral effect observed. As a comparison, the previously reported essential oil of Satureja montana was an inhibitor of TMV and CMV with antiviral activity rates of 29.2% and 24.1%, respectively [21]. Dominant components of this oil were the oxygenated monoterpenes thymol and carvacrol, while the investigated oils from Teucrium species are rich in sesquiterpenes. Comparing the percentages of inhibition, essential oils of T. polium and T. montanum showed significantly stronger antiviral activity against CMV than essential oil of S. montana [21]. Other literature data dealing with antiviral activity of essential oils do not compare composition of oils and their antiviral effectiveness. Essential oil of Melaleuca alternifolia was previously reported as an inhibitor of TMV [13], while essential oil of Plectranthus tenuifloru showed an inhibitory effect against Tobacco Necrosis Virus, Tobacco Mosaic Virus and Tomato Spotted Wilt Virus [17]. Our conclusion is that sesquiterpene-rich essential oils are potent inhibitors of CMV infection and natural substances with possible role in the control of plant virus diseases. RI-identification by comparison to literature [24] and/or homemade library; MS-identification by NIST02 and Wiley 7 spectral databases; Co-GCidentification confirmed with reference compound; tr-traces (mean value below 0.1%); -= not identified.

Plant Material
Plant material was collected, as stated in Table 3

Isolation of Essential Oils
Aerial parts of plants were dried in a shady place at room temperature for 10 days. Plant tops during flowering were used for the analysis of essential oil composition. Dried aerial parts of plant material (100 g) were subjected to hydrodistillation for 3 h in a Clevenger type apparatus. The obtained essential oil was dried over anhydrous sodium sulphate.

Data Analysis and Data Evaluation
The individual peaks were identified by comparison of their retention indices (relative to C 8 -C 40 n-alkanes for VF-5MS and CP Wax 52) to those from a homemade library, literature [24] and/or authentic samples, as well as by comparing their mass spectra with literature [24], Wiley 7 MS (Wiley, New York, NY, USA) and NIST02 (Gaithersburg, MD, USA) mass spectral databases. The homemade library was created from authentic compounds obtained commercially and from the main components of many essential oils obtained during our previous studies. The component percentages were calculated as mean values form the GC and GC-MS peak areas using the normalization method (without correction factors).

Plant Host for Antiphytoviral Studies
Seeds of Chenopodium quinoa Willd. were sown in trays containing Klasmann universal compost and maintained in a growth chamber (26 °C, 16:8 h light/dark cycle) with watering as required. When the seedlings were large enough to handle they were transplanted individually into perforated styrofoam which contained fresh compost for ten days and then into 10 cm plastic pots with fresh compost. Plants were grown in a growth chamber under same conditions. Experimental plants were selected three to 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.

Solution of Essential Oil
Spray solution containing 500 ppm of essential oil in distilled water was prepared for testing the antiphytoviral effect. To overcome insolubility of the oil in water 0.05 mL of Tween 80 was mixed with 25 µL of the oil and 50 mL of distilled water. Prepared solution was immediately sprayed to the test plants.

Viral Inoculums
Viral inoculum was prepared from leaf tissue obtained from Nicotiana megalosiphon Van Heurck & Muell. Arg. plants previously infected with Cucumber Mosaic Virus (CMV). Leaves of systemically infected leaf material (3)(4)(5) were ground with 10 mL of inoculation buffer (0.06 mol/L phosphate buffer, pH = 7.0) in a mortar. Sap extract was diluted with the same inoculation buffer to give a suitable number of discrete local lesions on test plants. The inoculum prepared in this way was used to inoculate local host Chenopodium quinoa.

Application to the Local Host Plants
Spray solution of essential oil was applied to Chenopodium quinoa plants for two successive days prior to CMV inoculation. Control spray was of distilled water and Tween 80. Plants were sprayed with either an oil or control solution. The second day of treatment plants were left to dry for 20 min and then inoculated with prepared viral inoculum. All treatments were repeated for three times on plants selected for uniformity and grown in a growth chamber (26 °C; 16:8 h light/dark cycle). Local lesions were counted 6th day post inoculation and the inhibition percentage was calculated by comparing the number of viral lesions on treated and control group according to the formula: where IP = antiviral inhibition in %, CK = average number of viral lesions on the control group; A = average number of viral lesions on the essential oil treated group.

Statistical Analysis
The significance of difference between mean value for treatment and control was estimated statistically using one tailed Student t-test (GraphPad InStat software).

Conclusion
Our previous investigations as well as the investigation carried out in this study confirmed that essential oils are potent antiphytoviral agents. Based on the limited number of publications published so far in this field, it could be concluded that there is no universal mode of antiphytoviral action of essential oils. β-Caryophyllene, the main component of the investigated Teucrium oils, and known to be effective antiphytoviral agent, is responsible for the observed antiviral activity. However it is also possible that some other components, which may be present in very small quantities, contribute to its effectiveness. Some of the oil components are already known as antiphytoviral agents, but many others must be tested to answer the question about the antiphytoviral mechanism of essential oils.