Composition of the Essential Oil of Salvia ballotiflora (Lamiaceae) and Its Insecticidal Activity

Essential oils can be used as an alternative to using synthetic insecticides for pest management. Therefore, the insectistatic and insecticidal activities of the essential oil of aerial parts of Salvia ballotiflora (Lamiaceae) were tested against the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae). The results demonstrated insecticidal and insectistatical activities against this insect pest with concentrations at 80 µg·mL−1 resulting in 20% larval viability and 10% pupal viability. The larval viability fifty (LV50) corresponded to a concentration of 128.8 µg·mL−1. This oil also increased the duration of the larval phase by 5.5 days and reduced the pupal weight by 29.2% withrespect to the control. The GC-MS analysis of the essential oil of S. ballotiflora showed its main components to be caryophyllene oxide (15.97%), and β-caryophyllene (12.74%), which showed insecticidal and insectistatical activities against S. frugiperda. The insecticidal activity of β-caryophyllene began at 80 µg·mL−1, giving a larval viability of 25% and viability pupal of 20%. The insectistatic activity also started at 80 µg·mL−1 reducing the pupal weight by 22.1% with respect to control. Caryophyllene oxide showed insecticidal activity at 80 µg·mL−1 giving a larval viability of 35% and viability pupal of 20%.The insectistatic activity started at 400 µg·mL−1 and increased the larval phase by 8.8% days with respect to control. The LV50 values for these compounds were 153.1 and 146.5 µg·mL−1, respectively.


Introduction
The caterpillar of Spodoptera frugiperda (Lepidoptera: Noctuidae) is a significant polyphagous insect pest of agricultural importance, not only for the damage it causes, but also due to its control difficulties [1]. This species inhabits the American continent from southern Canada to Argentina and causes considerable economic losses in several important crops such as maize, sorghum, rice, cotton, alfalfa, forage grasses, and occasionally other crops in the majority of the countries within its range [2][3][4]. The principal method to control this insect is the application of synthetic insecticides which yield effective results over a short time period, however, resistance development to these insecticides is also very fast, leading farmers to increase dosages or change the active ingredient frequently [5][6][7].
The implementation of Integrated Pest Management Programs appear to be a suitable alternative to managing noctuid insects by combining different methods that include the use of botanical extracts as insecticides [8]. Due to this, there has been a growing interest in botanical resources with activity against insect pests. This need has originated from the demand to provide alternatives to reduce the use of synthetic insecticides, which can have adverse effects on the environment [9]. The use of essential oils against insect pests has been frequently proposed. According Koul et al. [10] these substances are defined as any volatile oil(s) that have strong aromatic components and that give a distinctive odor, flavor or scent to a plant. These oils can be found in glandular hairs or secretory cavities of plant-cell walls and are present as droplets of fluid in the leaves, stems, bark, flowers, roots, and/or fruits of different plants. The essential oils provide various functions for the plants including: (i) attracting or repelling insects; (ii) protecting them from heat or cold; and (iii) utilizing chemical constituents in the oil as means of defense. Many plant essential oils obtained from Salvia species and their constituent compounds have been evaluated against insect pests and a number of them have shown considerable promise for the development of natural repellents/insecticides [11][12][13][14]. The Salvia genus is the most diverse of Lamiaceae Family, with over 1000 species around the world distributed in tropical and subtropical zones. In Mexico, there are at least 300 species reported [15]. This genus demonstrates an affinity to pine and oak forests as well as cloud and tropical deciduous forest, but also has shown remarkable diversity and endemism in arid and desert zones. The Mexican states richest in these species are: Oaxaca, Guerrero, Puebla, Jalisco, Michoacán, Coahuila, Baja California Sur, Tamaulipas and San Luis Potosí [16]. Therefore, the aim of this study was to determine insecticidal and insectistatic activities against S. frugiperda of the essential oil of the aerial parts of Salvia ballotiflora (Lamiaceae) and its main components.

Plant Material
The aerial parts (leaves, stems and flowers) of S. ballotiflora were collected in the Municipio of Guadalcázar, San Luis Potosí, México, at 1640 m.a.l.s., in September of 2013, the Taxonomic authentication was performed by José García-Pérez at the Isidro Palacios Herbarium of the Universidad Autónoma de San Luis Potosí. A voucher specimen was stored (SLPM 43013).

Essential Oil Extraction
Aerial parts of the plant weighing approximately 2 kg, were submitted to hydrodistillation for 3 hours. The mixture obtained was treated with ethyl ether, then the organic phase was separated and concentrated with a rotatory evaporator at 18 °C. The essential oil obtained was dehydrated with anhydrous sodium sulphate, and the ethyl ether residue was eliminated under vacuum, to give a yellow amber essential oil with a density of 0.6836 g·mL −1 at 20 °C, and refraction index of 1.4095 at 25 °C. The yield was (0.47 w/w). The oil was protected from direct light and stored at 4 °C until its use.

Identification of Essential Oil Main Components
S. ballotiflora essential oil samples (20 µL) were diluted with acetone (1 mL). The essential oil was analyzed on an Agilent Technologies (Santa Clara, CA, USA) 6890N GC equipped with an HP-5MS column (30 m in length; 25 mm internal diameter; 0.25 μm film thicknesses) and an Agilent EM 5973 detector, at 250 °C. The carrier gas was helium, with a flow rate of 1 mL·min −1 ; the split ratio was 2:1. The column temperature was initially 50 °C (for 3 min) and was gradually increased to 240 °C, at 3 °C·min −1 ; this temperature was held for 2 min. The injector temperature was 250 °C and 1 μL of essential oil was injected as a duplicate. The spectra were collected at 71 eV ionization voltages and the analyzed mass range was 15-600 m/z. The identification of the components were confirmed by comparison of the retention indices with those of authentic compounds using the Kovats index, based on n-alcanes C6-C26 with the Wiley09/NIST11 library. The identification of the two main components was confirmed by comparison of the retention indices to those of authentic compounds.

Insect Rearing
Fall armyworm (S. frugiperda) larvae were reared in the Insecticide Natural Compounds Laboratory from the Chemistry Faculty of the Autonomous University of Querétaro, according to the Bergvinson and Kumar [35] methodology using the following parameters: temperature 25 ± 2 °C with a relative humidity of 70% and 12/12 h light/dark cycles. For 1 kg diet feed for S. frugiperda, the following ingredients were used: 800 mL of distilled water, 60 g diet (Product# F0635 S.W. Corn Borer, Bio-Serv, Frenchtown, NJ, USA), 20 g sterile corn spike, 100 g ground corn, 40 g brewer's yeast, 10 g vitamins (vitamin mix fortification lepidoptera, Bio-Serv), 10 g agar, 1.7 g sorbic acid (dissolved in the ethanol), 17 mL ethanol, 2.5 mL formaldehyde, 1.7 g methyl p-hydroxybenzoate and 0.6 g neomycin sulfate.

Bioassay
For the bioassay, first instar larvae of S. frugiperda were used. Groups of 20 larvae were randomly selected for each concentration of S. ballotiflora essential oil, β-caryophyllene and caryophyllene oxide. Preliminary screening of essential oil for each compound was carried out at five concentrations (0.1, 1, 10, 100 and 1000 µg·mL −1 ). Based on the preliminary screening results, the concentration-dependent levels were selected (80, 160, 400, 600 and 1000 µg·mL −1 ). The test included a negative control (diet only). The essential oil, β-caryophyllene and caryophyllene oxide were mixed with the larvae diet ingredients during preparation without use of any solvent, according the methodology of Ramos-López et al. [36]. The effect of essential oil and the compounds were monitored during all larval stages "known as the larval-phase duration", and the pupal stage "called the pupal-phase duration". The number of pupae formed (larval viability), number of adults formed (pupal viability), and weight of pupae at 24 hours were assessed. The larval viability (LV50) corresponded to 50% of the larvae of fall armyworm during all larval phases for each extract.

Statistical Analysis
Statistical analysis was conducted and data was assessed for normality and homoscedasticity prior to analysis. In some cases Kruskal-Wallis non-parametric analysis of variance was used when data violated these assumptions and could not be corrected using a transformation. ANOVA analysis and Tukey test were also performed, and the LV50 were calculated by Probit analysis, using the SYSTAT statistical analysis program [37].

Conclusions
The essential oil of aerial parts of S. ballotiflora had insectistatic and insecticidal activities against S. frugiperda. β-caryophyllene and caryophyllene oxide were the main components of S. ballotiflora essential oil and these compounds also showed insectistatic and insecticide activities against the fall armyworm.