Acaricidal activities of Santolina africana and Hertia cheirifolia essential oils against the two-spotted spider mite

BACKGROUND: Many plant essential oils show a broad spectrum of activity against pests. This study investigated the effects of two essential oils on Tetranychus urticae, one of the most serious pests in the world.

RESULTS: The chemical composition of the two oils was characterised by GC-MS. The most abundant component in the Santolina africana (Jord. & Fourr) oil was terpinen-4-ol (54.96%), while thymol (61%) was prevalent in the Hertia cheirifolia (L.) oil. Mortality and fecundity were measured upon treatment with oil concentrations ranging from 0.07 to 6.75 mg L^-1 with a Potter spray tower. Mite mortality increased with oil concentration, with LC₅₀ values of 2.35 mg L^-1 for S. africana and 3.43 mg L^-1 for H. cheirifolia respectively. For both oils, a reduction in fecundity was observed at concentrations of 0.07, 0.09 and 0.29 mg L^-1. Artificial blends of constituents of oils were also prepared and tested with individual constituents missing from the mixture. The results showed that the presence of all constituents was necessary to equal the toxicity of the two natural oils.

CONCLUSION: S. africana and H. cheirifolia oils can provide valuable acaricide activity with significantly lower LC₅₀ values. Thus, these oils cause important mortality and reduce the number of eggs laid by females. © 2012 Society of Chemical Industry

Plants have long been used by many societies throughout the world to kill or repel pests. Indeed, plants would be extinct if they had no defence against insects and other arthropod pests.¹ Plants produce chemicals that function as defence mechanisms to reduce feeding injury caused by phytophagous organisms. These natural chemicals have multiple modes of action, including antifeedant and repellent activities, moulting and respiration inhibition, growth and fecundity reduction and cuticle disruption.^2-8 These multiple modes of action are advantageous because they delay the development of resistance among arthropod pest populations.⁹ In addition, most essential oil chemicals are relatively non-toxic to mammals and fish in toxicological tests, and their low toxicity to humans make them attractive for use in pest control.¹⁰ For example, essential oils have shorter residual activity and are less persistent than conventional pesticides because they tend to be volatile and are susceptible to temperature and UV light degradation.^11,12 Because of these properties, essential oils from aromatic plants have been widely investigated in the search for plant-derived chemical alternatives to conventional pesticides. In fact, previous investigations have demonstrated mite repellent effects for several different essential oils.^13-20 However, the efficiency of essential oils may depend on numerous factors, including the precise exposure concentration during application, highlighting the importance of conducting efficiency analyses for plant-derived pesticides over a wide range of concentrations.

Since 1900, the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) has become an increasingly important agricultural pest.²¹ It is a worldwide pest of many plant species, including several economically important agricultural crops.²² In fact, the wide ecological range of this mite is best reflected by the large diversity of host plants that the species exploits: to date, a total of 3877 host species have been reported around the world.²³

T. urticae is usually controlled by application of synthetic acaricides,^23-27 and integrated T. urticae pest management is largely dependent on the use of a few selective acaricides.²⁸ However, management of T. urticae is becoming increasingly difficult, because some populations have developed resistance to the chemicals used for their control.²⁹ Thus, the use of chemical insecticides is becoming increasingly inefficient and is associated

with significant ecological concerns. Therefore, it is imperative to develop other methods to prevent, or at least limit, the extent of damage from T. urticae.²⁰

Synthetic acaricides usually contain a single active compound; however, botanical pesticides such as plant essential oils are complex mixtures of several constituents. Previous studies reported the acaricidal activities of essential oils and their major constituents against T. urticae.^8,12,31 Indeed, many plants, including garlic (Allium sativum L.), rosemary (Rosmarinus officinalis L.) have been used to control this pest.^3,12,29

In the present study, plants were selected on the basis of their local use as insect controllers or insecticides. S. africana and H. cheirifolia (Asteraceae) are both alternatives to synthetic chemical pesticides and are widely distributed in northern and central Tunisia.^30,32,33

Therefore, the purpose of the present study was to evaluate the effectiveness of Santolina africana Jord. & Fourrand Hertia cherifolia L essential oils on the fecundity and mortality rates of T. urticae as a function of extract concentration. In addition, because little data were available in the literature on the bioactivities and chemical characteristics of S. africana and H. cheirifolia, these plants were analysed directly to determine which compounds could be responsible for their observed effects. Afterwards, to corroborate the role of each constituent in the toxicity to T. urticae, we reconstituted an artificial blend based on the proportion of the different compounds in natural oils but one and that for each of the major constituents.
2 MATERIALS AND METHODS

2.1 Chemicals

Pure compounds were purchased from Sigma Aldrich (St Louis, MO). Purities of these compounds varied from 95 to 99%.

Aerial parts (leaves and stems) of S. africana and H. cheirifolia used for this study were collected locally in Tunisia (Téjrouine, Sud West Kef) in June 2010 and were free of any preharvest chemical treatments (organic products). Plant samples were freshly harvested, sorted for uniformity and inspected to ensure the absence of defects. Plant samples were stored at -2°C until analysis.

The essential oil yield of the plants used in the present study was 0.5% of the dry weight of S. africana and 0.7% of H. cheirifolia; however, the colour of the two oils is clear and non-viscous.

GC-MS analyses of essential oils from S. africana and H. cheirifolia extracts were conducted in the Department of General and Organic Chemistry at Gembloux Agro-Bio Tech (University of Liège, Belgium).

Conventional GC/MS analyses were conducted using an Agilent GC 7975 coupled with an El mass selective detector (Agilent, Diegem, Belgium) and equipped with an HP5-MS capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness).⁸ The oven temperature programme was initiated at 40°C, held for 5 min at this temperature, then raised at a rate of 6°C min^-1 to 120°C, held for 5 min and then raised in a second ramp at a rate of 8 °C min^-1 to 300 °C. Helium was used as a carrier gas at a constant flow rate of 1 mL min^-1. An injection volume of 1 µL was used in splitless mode. The injection temperature was 250°C. MS detection was performed in electron impact (EI) mode at 70 eV by operating in full-scan acquisition mode in the 40-550 amu range. Volatile compounds were identified by comparing obtained mass spectra with those from the Wiley 275 L spectral library and with their retention indices.

Retention indices were determined relative to the retention times of a series of n-alkane standards (C9-C30, Sigma-Aldrich, 0.025 µg µL^-1 in n-hexane) measured under the chromatographic conditions described above, and were compared with literature values.³⁴

Fast GC analyses were conducted on a Thermo Ultra-Fast Trace GC gas chromatograph operated with a split/splitless injector and a Thermo AS 3000 autosampler (Thermo Electron Corp.). The GC system was equipped with an ultrafast module (UFM) incorporating a direct resistively heated column (Thermo Electron Corp.): UFC-5,5% phenyl, 5 m × 0.1 mm ID, 0.1 µm film thickness. The following chromatographic conditions were used to obtain a suitable peak resolution. The UFM temperature programme was as follows: initial temperature at 40°C, held for 0.1 min, ramp 1 at 30°C min^-1 to 95°C, ramp 2 at 35°C min^-1 to 155°C, ramp 3 at 200°C min^-1 to 280°C, held for 0.5 min.The injection temperature was 240°C, the injection volume was 1 µL, the carrier gas was He at a constant flow rate of 0.5 mL min^-1 and the split ratio was 1:100. The GC unit had a high-frequency fast flame ionisation detector (300 Hz FID). The temperature was 250°C, the H₂ flow rate was 35 mL min^-1, the air flow rate was 350 mL min^-1 and the make-up gas (N₂) flow rate was 30 mL min^-1. Data processing was performed using Chromcard software (v.2.3.3).

• Screening acute toxicity. A group of 25 T. urticae females (24 h old) were randomly selected and transferred to fresh bean leaf discs (35 mm diameter) placed adaxial side up on moistened cotton in petri dishes (90 × 15 mm) and were exposed to 12 different concentrations of oils (0.07,0.09,0.29,0.74,1.49,2.25, 2.99,3.74,4.5,5.24,5.99 and 6.75 mg L^-1 ). A Potter spray tower, which produces a deposit following a settling time of 1.5 min, was used to spray the mites on the leaf discs at a pressure of 1.4 bar at 20 ± 2°C. The distance of the sprayer nozzle from the leaves was 50 cm. Tests were repeated 5 times per concentration. Control tests were performed using water and ethanol (6:1). The bean leaf discs were maintained at room temperature (26 ± 1 °C, 60 ± 10% RH) at a photoperiod of 16:8h(L:D).

• Effect of essential oil on mortality. Mites were considered to be dead if their appendages did not move when prodded with a fine pencil 72 h post-treatment. For each concentration, the mortality rate (mean number of deaths post-treatment minus the mean number of deaths in the control) divided by the total number of females at the beginning of the tests (25 females) was calculated.^7,8 Each treatment was repeated 5 times, and the mean of the number of deaths was calculated from the five repetitions.^7,8 A test solution of a mixture (1:1) of the two oils was also studied. The distribution of mortality rates was best fitted using a sigmoidal curve (probit analysis).

• Effect of constituents of essential oils on mortality. The toxicity experiments were repeated with commercially available constituents of the essential oil and with blends of these oils with the same proportion as reported in Tables 1 and 2. To identify the relative contribution of each constituent to the toxicity of these oils, artificial blends of each of the two oils were made, including all major constituents of each oil called a 'full mixture' (FM for S. africana oil and TM for H. cheirifolia oil). Then the full mixture was compared with artificial blends, each missing one constituent (Figs 1 and 2). This classification of toxicity was similar to that used by Miresmailli et al.¹²

Sublethal concentrations (0.07, 0.09, 0.29 mg L^-1) were used to test the effect of these oils on female fecundity (25 females). After spraying, each female was transferred to a bean leaf disc (diameter 15 mm) to check for fecundity. The number of eggs laid by treated females was recorded for a period of 12 days before being destroyed. The number of eggs was best fitted to a sigmoidal curve (GraphPad Prism, Copeland, 2000), using the formula

where Y represents the value of the cumulative number of eggs at age X, K' is equal to the inflection point when h = 1, M is the maximum number of eggs (plateau value) and h represents the slope.^7,8 The fecundity of females treated with 0.07, 0.09 and 0.29 mg L^-1 essential oils was compared with a solvent (water and ethanol) control.

All the data were corrected using Abbott's formula. The LC₅₀, LC₉₀ and LC₁₀₀ values were determined by probit analysis using the Statplus program v.2009 (AnalystSoft Inc.). Tests were performed using one-way analysis of variance (ANOVA), and Newman-Keuls tests were used to compare means using Graph Pad Prism v.5.01 for Windows (GraphPad Software, San Diego, CA, http://www.graphPad.com). All tests were applied under the two-tailed hypothesis, with the level of statistical significance P set at 0.05.

The chemical compositions of the two essential oils evaluated in this study are shown in Tables 1 and 2. Experimental retention indices were compared with literature values,³⁵ and El mass spectra from each peak were compared with the library. Using this approach, it was possible to identify 12 individual components from S. africana and nine components from H. cheirifolia, representing 95.46 and 93.09% of the total weight respectively.

The two oils differed in their most abundant components. The most abundant components in the Santolina oil were terpinen-4-ol (54.96%), α-terpineol (14%) and borneol (8.37%) (Table 1), whereas thymol (61%) and 2,6-dimethoxy-phenol (12.83%) were most prevalent in the Hertia oil sample (Table 2).