4.4. Bioassays of Cyflumetofen- and Bifenthrin-Resistant Strains of T. urticae

Owing to the phenomenon of the mites escaping into the surrounding water after cyflumetofen treatment, the slide dip method was used to measure the mortality of the cyflumetofen-resistant strain [46]. Specifically, a 2 × 2 cm piece of double-sided adhesive tape was attached to one side of glass microscope slides, and then, 30 adult female mites were affixed to the tape by their dorsal surface. Note that the feet and mouthparts of mites should not be stuck to the tape. The glass microscope slides were placed in a tray covered with wet cotton and then incubated at 26 °C. After 4 h, the dead or inactive individuals were removed and new active female adults were supplemented. Then, five concentrations of cyflumetofen obtained by diluting cyflumetofen solution with water and 40% acetone and the control (i.e., water and 40% acetone) were used for testing. Each concentration had three replicates. Slides were dipped into the solution of cyflumetofen for 5 s. Then the slides were taken out and placed on a paper towel to dry. The extra solution was moved from the slides with filter paper. The treated slides, lined with slightly moistened paper towels, were left in the plastic tray with a cover and incubated under the condition of 16:8 (L:D) h at 25 ± 1 °C with 50–60% RH. The mortality criterion used for this method was the inability to move a leg when lightly prodded. After 24 h, the mortality of the cyflumetofen-resistant strain was determined using a microscope.

The mortality of the bifenthrin-resistant strain was measured according to the leaf residual toxicity method recommended by the FAO [46]. Briefly, five concentrations (with a 2-fold increase between concentrations) of bifenthrin were obtained by diluting the stock solution of bifenthrin (dissolved in acetone) with 0.1% Tween water and 40% acetone. The control contained 0.1% Tween water and 40% acetone. Then, cowpea leaf discs (4 cm in diameter) were dipped into the solution of bifenthrin for 10 s and subsequently placed on Petri dishes filled with water to dry naturally. Furthermore, 20 adult females of T. urticae were gently transported to each of the leaves using a soft brush. The petri dishes were kept in incubators under a cycle of 16:8 (L:D) h at 25 ± 1 °C and 50–60% RH. After 48 h, the mortality of the cyflumetofen-resistant strain was determined using a microscope. The mortality criterion was the same as the abovementioned.

The LC₅₀ value was determined by the corrected mortality calculated with Abbott’s formula. The bifenthrin-resistant strain (R_bft) and the cyflumetofen-resistant strain (R_cfm) were cultivated following the abovementioned process. The resistance ratio (RR) was calculated as the following [47]: