Imidacloprid

Spring wheat cultivar AC Fielder and AC Andrew were grown in a growth cabinet with a photoperiod of 18 h per day (intensity 300 μE m^–2 s^–1), as mentioned in Sinha and Eudes (2015) (link). The plants were treated with 2.5 ml l^–1 Tilt^TM (propiconazole, Syngenta) before the tillering stage (Zadok et al., 1974 (link)) and Intercept^TM (0.004 g/L of soil, Imidacloprid, Bayer) once sufficient root development was established. The first seven tillers from each pot were harvested when microspores were in the mid to late uninucleate stage; the microspore stage was verified from a median floret using acetocarmine staining (Alexander, 1969 (link)). The remaining spikes were allowed to grow half their length out of the boot and then harvested for ovaries. Wheat spikes for microspore isolation were kept in the refrigerator (4°C) for 3 weeks with their bases in distilled water and their heads wrapped in aluminum foil. After 3 weeks ± 3 days, the spikes were extracted from the tillers and the most homogenous spikes were used for the microspore extraction.

The pesticides were purchased as three commercially available formulations (suspension concentrate; SC, water-dispersible granule; WG, and emulsifiable concentrate; EC). Diniconazole (SC, 5% active ingredient) was purchased from Dongbang agro Co., Ltd. (Seoul, South Korea). Chlorfenapyr (SC, 10% a.i.) was purchased from Shin Young agro Co., Ltd. (Seoul, South Korea). Imidacloprid (SC, 8% a.i.) was obtained from Bayer Crop Science (Seoul, South Korea). Thiamethoxam (WG, 10% a.i.), chlorantraniliprole (SC, 2.7% a.i.), fludioxonil (SC, 20% a.i.), and lufenuron (EC, 5% a.i.) were purchased from Syngenta Korea (Seoul, South Korea). Indoxacarb (WG, 5% a.i.) was purchased from Hanearl Science (Gangwon-do, Taebaek-si, South Korea). Azoxystrobin (SC, 21.7% a.i.) and pyraclostrobin (SC, 20% a.i.) were purchased from Chunjiinbiotec (Gangwon-do, Taebaek-si, South Korea).

A neat standard of imidacloprid (CAS 138261-41-3, 98.8% pure), thiamethoxam (CAS 153719-23-4, 95.2% pure), clothianidin (CAS 210880-92-5, 99.6% pure), imidacloprid-olefin (CAS 115086-54-9, 97.9% pure), and 5-OH-imidacloprid (CAS 380912-09-4, 96.7% pure) were received as a gift from Bayer CropScience (Research Triangle Park, NC, USA). Deuterated internal standards clothianidin-d3 and thiamethoxam-d3 were obtained from Sigma-Aldrich (St. Louis, MO, USA). imidacloprid olefin-¹³C3,¹⁵N and imidacloprid-pyr-d₄-methyl-d₂, ¹³C were received from Bayer CropScience, and 5-OH-imidacloprid-¹³C,¹⁵N was received from Clearsynth (Mississauga, Ontario, Canada). Organic solvents (Optima LC-MS grade methanol, water, and acetonitrile), ammonium formate (99% pure), and 99% pure formic acid were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Fifty-milliliter QuEChERS tubes (part number 60105-216) and dispersive solid-phase extraction (dSPE) equipment (part number 60105-202; 60105-222; 60105-223) were obtained from Thermo Fisher Scientific.

Chlorantraniliprole (10 gL^-1 Coragen®; Du Pont), Imidacloprid (200 gL^-1 Confidor®; Bayer Crop Science), and Spinosad (10 gL^-1 Success®; Yates) were all purchased commercially and diluted to 5,000ppm stocks using distilled water. On the day of exposure, 5x stocks were generated for each dose being used (Chlorantraniliprole: 60, 30, 15, 7.5, 3.75, 0ppm; Imidacloprid: 240, 120, 60, 30, 15, 0ppm; Spinosad: 240, 60, 30, 15, 7.5, 3.75, 0 ppm) by diluting the 5,000ppm stock in 5% Analytical Reagent sucrose (Chem Supply). A similar procedure was followed for Ivermectin (Sigma), and a 10,000ppm stock was generated using DMSO as a solvent. 5x stocks were generated (120, 60, 30, 15, 7.5, 0 ppm) by dissolving the original stock in 5% sucrose, and the highest concentration of DMSO used in dosing was added to the 0ppm solution in order to control for solvent effects, none of which were observed.

Technical grade imidacloprid (99.4%) was provided by Bayer CropScience Canada, Inc. (Guelph, ON) and was dissolved in analytical grade acetone for topical applications.

Whitefly colonies (Bemisia tabaci, biotype B), reared on cotton plants (Gossypium hirsutum), were used for TYLCV (Genbank accession No. X15656) inoculation as described [23 , 35 ]. Thereafter, plants were sprayed with imidacloprid (Bayer, Leverkusen, Germany) and held in an insect-proof greenhouse at 26–32°C. TYLCV-induced symptoms were evaluated for each plant separately at 28 and 42 DPI according to a disease severity index (DSI) of 0-to-4 as follows: (0) no visible symptoms, inoculated plants show same growth and development as non-inoculated plants; (1) very slight yellowing of leaflet margins on apical leaf; (2) some yellowing and curling of leaflet ends; (3) a wide range of leaf yellowing, curling and cupping, with reduction in size, yet plants continue to develop and (4) very severe plant stunting and yellowing, pronounced cupping and curling, plants stop growth [21 , 24 ].