Laboratory Bioassay
Range-finding studies were run to determine the appropriate concentrations of the clove essential oil and its constituent compounds for determining LD50 values. Five concentrations of clove essential oil and its two constituents (eugenol and β-caryophyllene) were selected for adults (0.25–4.00 mg/ml) and nymphs (0.95–15.20 mg/ml). Five concentrations of eugenol acetate were used for adults and nymphs test at the range of 2.35–37.60 mg/ml. Five concentrations of abamectin (0.01–0.16 mg/ml) were used for both the adults and nymphs as a positive control, and acetone alone was used as a negative control. Five replicates were prepared for each dose level.
Petri dishes (9 cm diameter) were prepared with a wet paper disc and a fresh 8 cm diameter pear leaf. Groups of 10 third-fifth instar nymphs of C. chinensis were transferred directly into a prepared Petri dish by a fine camel’s hair brush as one replication. The adults were anesthetized by ether and transferred into a glass Petri dish which was placed on ice. An aliquot (0.5 µl) of oil or each component dilution was applied topically to the dorsal thorax of nymphs and adults using Burkard Arnold micro applicators (Burkard Scientific Supply, Rickmansworth, UK). Then the treated nymphs were kept in the prepared Petri dish. Groups of 10 treated adults were transferred into a prepared Petri dish as one replication as well.
All the Petri dishes were put into an incubator with 28°C, 90% RH, and a long-day photoperiod of 15:9 (L:D). Mortality was recorded after 24 h. Adults and nymphs were considered dead if they failed to respond when probed with a fine camel’s hair brush.