Foliar application effects of salicylic acid and jasmonic acid on the essential oil composition of Salvia officinalis [Salvia officinalis’in uçucu yağ bileşimi üzerinde Salisilik Jasmonik asidin

Aim: In this research, the effects of two elicitors [jasmonic acid (JA) and salicylic acid (SA)] on the accumulation of essential oils in the seed cultures of Sage ( Salvia officinalis L.) were studied. Materials and methods: This research was conducted at the research field of Islamic Azad University, Shahrekord branch located at 50 ° 56/E longitude, 32 ° 18/N latitude during 2015 and 2016. The study area was clas-sified as semi cold with an average temperature of 11.5 ° C and semi-arid with 329.9 mm of average rainfall. Seeds of sage were planted under field conditions. The following treatments were applied after the plants had four leaves: water, acetone, JA (0.1051, 0.2102, 0.4204, 0.8408 g/L), SA (0.0138, 0.138, 0.276, 0.552 g/L) and control. Results: Twenty-seven essential oils were obtained and the most notable were: α -pinene, limonene, β -pinene, camphor, thymol, camphene, thujone-trans, thujone-cis, 1,8-cineole, borneol, borneol acetate, carvacrol, α -humulene, caryophyllene. JA was more effective in stimulating the accumulation of α -pinene, limonene, β -pinene, camphor, thymol, camphene, thujone-trans, thujone-cis, 1,8-cineole, borneol, borneol acetate, carvacrol, α -humulene and caryophyllene. Conclusion: JA and SA had increasing effects on essential oils. The best treatments were found to be JA 0.1051 g/L, JA 0.2102 g/L and SA 0.138 g/L.


Introduction
Salvia officinalis (Lamiaceae) is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent, anti hidrotic and specific sensorial properties. Its essential oil is mainly composed of monoterpens. 1,8-cineole, thujone and camphor are responsible for some of these effects. The extraction of essential oil depends on genetic and environmental properties, as well as the process and fresh weight of the plant. The genus Salvia (Labiatae) contains more than 700 species of which about 200 exist in Iran. Plants belonging to this genus are pharmacologically active and have been used in folk medicine all around the world. Salvia seeds are small and round, with a mucilage layer on their surface which swells in water to give a viscous suspension [1,2].
Salicylic acid (SA) and jasmonic acid (JA) are signal molecules participating in the mechanism of plant resistance to related diseases, especially with the induction of pro-antioxidant system generation in plant tissues. Since both SA and JA improve plant resistance to diseases and improve plant productivity, this combination is applied in agriculture [3,4]. Salicylic acid, a naturally occurring plant hormone, acts as an important signaling molecule in plants, and has diverse effects on abiotic stress tolerance. The exogenous application of SA may be involved in the regulation of plant physiological processes such as stomata closure, ion uptake and transport, membrane permeability, as well as photosynthesis and growth [5]. Its role in abiotic stress tolerance and osmotic stress has been reported [6,7]. The phenolic compound of SA plays a vital role in the defense response against many pathogens which induce the expression of many defense related genes [8]. There was an increase in the process of tolerance to different stresses by SA [9], and the greatest effect was found in many elicitors [10]. Low levels of salinity result in a decrease in germination and have no effect on high levels of salinity [11]. SA increased due to resistance to stresses such as salt stress. The foliar application of this elicitor resulted in greater root, shoot and total dry weights of calendula plants under salt stress [12], decreased effects and damage of drought stress on germination and seedlings growth [1], increased photosynthetic pigments, NPK, Fe, Zn, Mn, total carbohydrates and crude protein concentrations in leaves [13]. SA reformed and detoxified super oxide radicals which yielded beneficial physiological and morphological effects and produced a salt tolerant variety [14]. Nutrients significantly increased the number of umbel per coriander (Coriandrum sativum L.) but SA had the opposite effect [15]. In pepper, SA increased growth and development [16]. Under Cu-stressed condition, SA induced the synthesis of polypeptides and translocation of Cu and/or increased Cu-binding proteins [17]. SA decreased polyphenol oxidase and peroxidase. It seems that SA can considerably alleviate the oxidative damage that occurs under cold stress condition. SA decreased the activity of antioxidant enzymes, reduced disease severity and increased the amount of secondary compounds in infected and non-infected daisy plants. SA failed to show any stimulatory effect on either cell growth or hypericin production [18]. The application of JA in stressed plants reduced the amount of lipid peroxidation, stimulated the synthesis of antioxidant enzymes, and enhanced the content and yield of artemisinin [3]. The JA elicitor (100 μM) enhanced total isoflavones production and stimulated the accumulation of daidzein and genistein in Puerarialobata [19]. JA reduced transpiration and membrane-lipid peroxidation as expressed by malondialdehyde in strawberry plants [20].
This research was conducted in 2 years, so as to evaluate the effects of the two elicitors [jasmonic acid (JA) and salicylic acid (SA)] on the essential oils content and composition in the seed cultures of Sage (S. officinalis L.).

Materials and methods
Sage (S. officinalis L.) seeds were obtained from the Iranian Seeds and Plant Improvement Institute, and planted in field condition. The following treatments were applied after the plants had four leaves: T 1 , water; T 2 , acetone; T 3 , JA 0.1051 g/L; T 4 , JA 0.2102 g/L; T 5 , JA 0.4204 g/L; T 6 , JA 0.8408 g/L; T 7 , SA 0.0138 g/L; T 8 , SA 0.138 g/L; T 9 , SA 0.276 g/L; T 10 , SA 0.552 g/L and control (without water, acetone, JA and SA). Field trials were established in 2015 and 2016 at Shahrekord (50°56/E 32°18/N) South Western Iran. The region has an elevation of 2060 m, annual precipitation of 329.9 mm, and average minimum, maximum and annual temperatures of −31°C, 27°C and 12°C, respectively. The soil (Typic calci xerocrepts) physical and chemical properties are shown in Table 1.
The topsoil of the experimental plot area was kept moist throughout the growing season when necessary. After soil test, the required nutrients were added to the soil. Seeds were sown in pots in the farm condition on 22 May, 2015 and 20 May, 2016.
Fresh aerial S. officinalis tissues were dried for 10 days at room temperature (25 ± 5°C), and then ground to fine powder using a Moulinex food processor. The essential oil was extracted by heating 50 g of leaf tissue in a 2 L flask with 1 L water, using a heating jacket at 100°C for 3 h in a Clevenger-type apparatus, according to the procedures outlined by the British Pharmacopeia. Voucher specimens (20071-TUH) were those described and deposited in the Herbarium of the Center of Agricultural and Natural Resources of Chaharmahal and Bakhtiari Province, Shahrekord, Iran.
The essential oil content was determined by distilling tissues in Clevenger type apparatus. One thousand gram of plant tissues were placed in 6 L Clevenger-type distillation apparatuses and distilled for 5 h in 3 L of pure water. The quantities of plant oils obtained at the end of distillation were measured in mL and ratios (% w/w) were determined by multiplying oil content with oil density (i.e. 0.858 g⋅cm 3 ). All the essential oil samples were dried over hydrous sodium sulfate, and stored at 4°C until GC and GC-MS analyses.
Ground GC analysis was done using Agilent Technologies 7890 GC equipped with FID and a HP-5MS 5% capillary column. The carrier gas was helium at a flow rate of 0.8 mL/min. The initial column temperature was 60°C and it was programmed to increase at 4°C/min to 280°C. The split ratio was 40:1. The injector temperature was set at 300°C. GC-MS analyses were conducted on a Thermo Finnegan Trace 2000 GC/MS, made in the USA, employed with a HP-5MS capillary column (30 m long and 0.25 mm wide, and a 0.25 μm of film thickness) using an injector chamber at a temperature of 250°C. Oven temperature was held at 120°C for 5 min and then programmed to reach 280°C at a rate of 10°C/min. The temperature of the detector and injector was 260°C. The composition of the essential oil was identified by comparing retention indices relative to a series of n-alkanes (C 7 -C 24 ); retention times and mass spectra were those of authentic samples from Wiley's library [21].
The experiments were arranged in a randomized complete block design with three replications, and 100 plants were used for each trial. All data were subjected to analysis of variance (ANOVA) using the statistical computer package SAS and treatment means were separated using L.S.D multiple range test at p < 0.05 level.

Results and discussion
Plant hormone treatment is a simple, eco-friendly and relatively commercially viable method of increasing the synthesis of phytochemicals; therefore, it may be used to improve the quality of the essential oil of sage plants.
Most studies have reported that JA and SA are two naturally occurring plant growth regulators involved in various aspects of plant development and response to biotic and abiotic stresses. The reports focused on their beneficial roles on defense mechanisms in stress conditions such as salinity, drought and pathogens. In this research, the role of these phytohormones on the accumulation of the important components in essential oil, was studied in 2 years. JA and SA had increasing effects on the essential oils. It was found that components of β-pinene, thymol, thujone-trans, thujone-cis, 1,8-cineole, borneol, by SA treatments and other components such as camphor, camphene, borneol acetate, and carvacrol, were significantly increased by JA treatments.
Various concentrations of elicitors produced different results. At low concentrations, SA did not have any inhibitory effect and made many components but at higher concentrations, it reduced the protein and essential oils content [22,23]. A concentration of 0.138 g/L of SA, had a stimulating effect while 0.138 g/L and 0.552 g/L concentration had varying degrees of inhibitive effects; therefore, SA had a bidirectional physiological effect in a concentrationdependent manner. JA and SA-treated plants produced greater quantities of camphene, 1,8-cineole, thujone-cis, thujone-trans, camphor, borneol, borneol acetate, carvacrol and thymol. The accumulation of phenolic compounds was stimulated with SA at low concentrations. In Salvia miltiorrhiza [24], mung bean [6], Caraway [25], Ziziphus spina-christi [26], Cucumber [16], Marigold [12] as well as Basil and majoram [5] also showed the decreasing effects  ns, a and b : Non significant, significant at the 5% and 1% levels of probability, respectively.   : Non significant, significant at the 5% and 1% levels of probability, respectively.   of a higher concentration of SA (foliar application) and otherwise showed its beneficial uses at low dosages. In this research, it was found that although some essential oils have a higher concentration of SA as compared with other treatments, but generally, the best SA treatment was 0.138 g/L. However, many researchers have shown that SA has various effects on plant growth and development [6,27,28] but the field application of SA requires optimum physiological concentration to increase nitrogen use efficiency, particularly during germination and seedling growth [29,30]. An increase in phenolic compounds and flavones was observed in cells, after JA elicitation. In contrast, anthocyanins were in lower amounts in JA treatment [31]. Similar with SA, JA has various effects on components. In Salvia miltiorrhiza [32], Brugmansia candida [19], Hypericum perforatum L. [31], Eleutherococcus senticosus [33], Sweet Basil [13], Kudzu [7], Panax ginseng [4], and Glycyrrhiza glabra [11], it was found that at low and high concentrations of JA, some components were either decreased or increased. Under water stress, JA increased total sugars, essential oil and major component and decreased the concentrations of total amino acids and proline [16,[34][35][36][37][38][39][40]. The results of this research are similar to the findings of Xiao et al. [32] and Dong et al. [24]. The research condition and many attributes such as the edaphic properties, affected the efficacy of JA.