Acaricidal activities of Santolina africana and Hertia cheirifolia essential oils against the two-spotted spider mite
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3.2 Toxicity of essential oils After 72 h, all mites in the control group were still alive. However, a few mortalities were observed at 0.07, 0.09 and 0.29 mg L-1 for both essential oils, and for the mixture of the two oils (Tables 3 and 4). For both S. africana and H. cheirifolia oils, female mortality increased with increasing concentration of essential oil. The experimental distribution of mortality rates was fitted with a sigmoid curve (R = 0.96, df = 64, N = 25, P < 0.0001; = 0.98, df = 64, N = 25, P < 0.0001 for H. cheirifolia and S. africana respectively). There was a significant difference between the three treatments-S. africana, H. cheirifolia and the mixture of both oils (F12,52 = 71.21, P < 0.001; F12,52 = 133.92, P < 0.001; F12,52 = 257.42, P < 0.001 respectively) - when comparing the different concentrations to control. Interestingly, a combination of oils from H. cheirifolia and S. africana provided better mite control (LC50 and LC90 values of 1.6 and 3.07 mg L-1) than each oil individually (2.35 and 3.79 mg L-1 for S. africana and 3.43 and 5.07 mg L-1 for H. cheirifolia respectively) (see Table 3).
The constituents of all oils were tested individually to assess their toxicity effects. A significant difference was found in the lethal toxicity of the single constituents of each oil when all treatments were compared: one-way ANOVA-F = 146.28; df = 14, 135; P < 0.0001 for S. africana (Fig. 3) and F = 195, 79; df = 11, 108; P < 0.0001 for H. cheirifolia (Fig. 4) respectively. Indeed, the Newman-Keuls tests comparing the toxicity of single constituents revealed that four constituents of S. africana (1,8-cineole, borneol, terpinen-4-ol and α-terpineol) were highly toxic, but that they were not as toxic individually as the essential oil and the full mixture. Four constituents of S. africana (camphor, trans-chrysanthenol, carvenone and carvacrol) were slightly toxic, whereas the remaining constituents (limonene, cis-jasmone, guaiol and α-bisabolo) were not toxic to. T. urticae not differ significantly from the mortality rate found in the control (Fig. 3). Four constituents of H. cheirifolia (1,8-cineole, terpinen-4-ol, α-terpineol and thymol) were highly toxic, but they were not as toxic individually as the essential oil and the full mixture. Four constituents of H. cheirifolia (camphor, para-cymen-8-ol, myrtenol and 6-methyl-alpha-ionone) were slightly toxic, whereas the remaining constituents (2,6-dimethoxy-phenol) were not toxic to T. urticae and did not differ significantly from the mortality rate found in the control (Fig. 4). 3.4 Toxicity of recomposed oils Based on the 100% lethal concentration (LC100 = 4.06 mg L-1 for S. africana and LC100 = 5.37 mg L-1 for H. cheirifolia), and following the natural composition of each oil as indicated by GC-MS (Tables 1 and 2), bioassays with recomposed oils for S. africana (limonene, 1,8-cineole, camphor, trans-chrysanthenol, borneol, terpinen-4-ol, α-terpineol, carvenone, carvacrol, cis-jasmone, guaiol,and α-bisabolol) and for H. cheirifolia (1,8-cineole, camphor, terpinen-4-ol, paracymen-8-ol, α-terpineol, myrtenol, thymol, 2,6-dimethoxy-phenol and 6-methyl-alpha-ionone) showed that the greatest mortality rate was obtained when all constituents were present (full mixture) (Figs 3 and 4). The mortality of these two full mixtures was significantly higher than the mortality rates for the most toxic constituents of each oil. Indeed, the mortality rate caused by the full mixture (a blend of 12 constituents of S. africana and nine constituents for H. cheirifolia) did not differ significantly to the mortality caused by the pure oil for each plant used in this study. 3.5 Toxicity of selected blends In the S. africana oil, component elimination assays indicated that the absence of 1,8-cineole and α-terpineol caused the largest decrease in toxicity of the blend. Missing of camphor, carvacrol, borneol, terpinen-4-ol, trans-chrysanthenol and carvenone resulted in significant decreases in mortality compared with both the oil and the full mixture, but less so than for 1,8-cineole and α-terpineol. The missing of any other constituent (limonene, c/s-jasmone, guaiol or α-bisabolol) did not produce any significant differences in mortality from either the pure oil or the full mixture (Fig.1). In the H. cheirifolia oil, 1,8-cineole, α-terpineol and thymol were found to contribute to the toxicity of the oil, whereas terpinen-4-ol, camphor, myrthenol and paracymen-8-ol had only a moderate influence on toxicity. The missing of the remaining constituents (2,6-dmethoxy-phenol or 6-methyl-alpha-ionone) had a low influence on toxicity (Fig. 2).
Fecundity was reduced following treatment with low concentrations of extract (Table 3). The experimental distribution of fecundity was fitted with a sigmoid curve one-way ANOVA (R = 0.78, df = 99, N = 25, P < 0.0001; R = 0.55, df = 99, N = 25, P < 0.0001; R = 0.80, df = 99, N = 25, P < 0.0001 for H. cheirifolia, S. africana and the mixture of the two oils respectively). Maximum values for the cumulative number of eggs were significantly reduced compared with the controls for sublethal extract concentrations (0.07, 0.09 and 0.29 mg L-1). There was a significant difference between the three treatments - S. africana, H. cheirifolia and the mixture of both oils (F3,96 = 39.53, P < 0.0001; F3,96 = 114.5, P < 0.0001; F3,96 = 135.8, P < 0.001 respectively) - when comparing the cumulative number of eggs to control.
In the present study, an examination was made of the effect of different concentrations of S. africana and H. cheirifolia essential oils on their efficacy as natural pesticides against an important arthropod pest, T. urticae. Both oils were found to cause significant mortality in T. urticae. Moreover, the effects of a wide range of essential oil concentrations were quantitifed: essential oil treatments caused significant T. urticae mortality at low concentrations, with LC50 values of 2.35 mg L-1 for S. africana and 3.43 mg L-1 for H. cheirifolia. In addition, the effects of individual essential oil treatments versus a mixture of the two oils on T. urticae fecundity were investigated. Although the lowest tested concentrations (0.07, 0.09 and 0.29 mg L-1) do not affect female survival rates, they do affect female fecundity versus the control group. Table 3. The effects of different concentrations of S. africana and H. cheirifolia essential oils on the percentage mortality of T. urticae adults. Each % mortality value is an average of five replicates ± confidence interval with 25 female adult mites per replicate. The estimated lethal concentration (LC50 and LC90) values (mg L-1) for each essential oil, the chi-square, the degree of freedom and the standard error were estimated using probit analysis. Mean percentages in any column are significantly different at P ≤ 0.05 (*) and P ≤ 0.001 (***); ns: not significant; Newman Keuls test
Interestingly, the present experimental results demonstrate that a combination of S. africana and H. cheirifolia oils provides better mite control than each oil alone. Previous studies of H. cheirifolia showed the presence of six sesquiterpenoid compounds: 8β-methoxy-10β-hydroxyere-mophilenolide, 10β-hydroxyeremophilenolide, 8β,10β-dihy-droxyeremophilenolide, bakkenolide A, the 6α,8/3-dimethoxy-10β-hydroxyeremophilenolide and 3β-angeloyloxy-10β-hydro-xyeremophilenolide.34,36,37 The activity of the S. africana oil may be explained by the presence of 1,8-cineole and α-terpineol, in spite of their low percentage in the oil. Their role as major contributors to toxicity was confirmed by missing 1,8-cineole (3.59%) and α-terpineol (14.06%) from the full mixture, leading to a significant decrease in the mortality caused by the oil (<20% mortality) (Fig. 2). However, in some instances, minor constituents were responsible for the overall activity of the oil. Missing of the major constituent terpinen-4-ol (54.96%) from the full mixture of S. africana produced a significant decrease in the mortality, but less than in the case of 1,8-cineole and α-terpineol. Similarly, the activity of H. cheirifolia oil may be attributed to the presence of 1,8-cineole, α-terpineol and thymol. In fact, their role as major contributors to toxicity was confirmed by missing 1,8-cineole, α-terpineol and thymol from the full mixture, leading to a significant decrease in the mortality caused by the oil (<20% mortality) (Fig. 2). In previous works, some other oils have demonstrated the same effect, where missing of major constituents has led to a significant decrease in toxicity of the oil.8,12,13 In many cases, missing of major constituents from the mixture led to a significant decrease in toxicity;12 for example, in Hertia oil, missing thymol (61%) from the full mixture produced a significant decrease in the mortality of T. urticae (<20% mortality); however, the missing of thymol did not differ significantly from the missing of 1,8-cineole and α-terpineol respectively. It can be inferred that thymol, 1,8-cineole and α-terpineol have the same effect against T. urticae. This indicates that the activity of some oils may be attributed to more than one compound, resulting in a synergetic effect between major compounds or minor compounds.8,12 The present results are in agreement with a previous report12 that 1,8-cineole is responsible for the major toxicity of rosemary oil against T. urticae. Understanding the role of each constituent to the overall activity of the oil provides an opportunity to create artificial blends that optimise their efficacy against different pests. One interesting aspect of the present study was the difference found in the role of the major constituents in a mixture as opposed to their individual toxicities; thus, the missing of the same compound from two different oil artificial mixtures produced the same proportion of mortality, in spite of the difference in their proportion in the pure oils. 1,8-Cineole constituted 3.59 and 1.29% of the artificial mixture of Santolina and Hertia oils respectively. Similarly, α-terpineol constituted 14.06 and 3.58% of the artificial mixture of Santolina and Hertia oils. In previous work, oxygenated monoterpenes, such as camphor, terpinen-4-ol and borneol, which were detected as major components in Salvia hydrangea oil, were shown to have antibacterial activity.38,39 In the present study, 1,8 cineole and α-terpineol were found to be the major constituent contributors to toxicity in Santolina oil. Similarly, 1,8-cineole, α-terpineol and thymol were the major contributors to the toxicity of Hertia oil. However, essential oils contain numerous components, and other major and/or minor compound(s) may also play a role in their acaricidal activity. The chemical composition of S. africana and H. cheirifoliα oils can provide valuable acaricide activity in the field, with significantly lower LC50 values (2.35 and 3.43 mg L-1) compared with other acaricides, such as fenbutatin oxide, which has an LC50 value of 20.6 mg L-1.31 The highest mortality rates were obtained in both oils when all the constituents were present in the mixture (>90% mortality). Knowing the role of each constituent in toxicity and the effects on fecundity of these oils makes it possible to create an artificial blend of different constituents on the basis of their activities and their effect on the pest. The quantitative composition of the essential oils of many aromatic plants is also greatly influenced by both the genotype and agronomic conditions, such as harvesting time, plantage and crop density.40 S. africana and H. cheirifolia provide an essential oil yield of <0.8%. Thus, although these oils provide good mortality and reduce the number of eggs laid by T. urticae females, their poor yields limit their large-scale applicability. Therefore, further development will be needed to implement these oils in pesticide applications, and more efficient methods of oil extraction and plant culture are required. Figure 3. Mortality caused by the oil, full mixture and individual constituents to T. urticae when applied at levels equivalent to those found in the 100% lethal concentration of the pure oil of S. africana (LC100 = 4.06 mg L-1). Error bars represent the standard error of the mean of ten replicates, each replicate containing 25 females. Means corresponding to each treatment with different letters are significantly different from each other (Newman -Keuls test, P < 0.05). 
Figure 4. Mortality caused by the oil, full mixture and individual constituents to T. urticae when applied at levels equivalent to those found in the 100% lethal concentration of the pure oil of H. cheirifolia (LC100 = 5.37 mg L-1). Error bars represent the standard error of the mean of ten replicates, each replicate containing 25 females. Means corresponding to each treatment with different letters are significantly different from each other (Newman -Keuls test, P < 0.05). 
Table 4. Comparison between the cumulative number of eggs laid by females treated with three different concentrations (0.07. 0.09 and 0.29 mg L-1) of S. africana and H. cheirifolia essential oils and with the control solution. Parameters: Top = the plateau value indicating the maximum number of offspring, or the number of eggs laid after 12 days. LT50 is the period of time (days) that provided a response half-way between 0 and the maximum number of eggs (plateau); h = the hill slope
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