2.3. Preparation and inoculation of J2s
SCN race 3 nematodes, which were originally collected from a field at the Southern Outreach and Research Station in Waseca, Minnesota, USA, were cultured on soybean plants in sterilized soil in a greenhouse. A soil suspension containing newly formed cysts was poured into a 2-liter jug and sprayed with a strong jet of water. This suspension was allowed to settle for 5–10 s and then poured onto a 850-μm-pore (#20) sieve nested on top of a 250-μm-aperture (#60) sieve. This procedure was repeated 5 times to ensure that all cysts were collected. The cysts were then sprayed with water on the 850-μm-aperture screen to remove root debris and collected onto a 250-μm-aperture sieve. The cysts were washed from the 250-μm-aperture sieve into a 50 mL centrifuge tube with 63% (w/v) sucrose and centrifuged at 1100×g for 5 min. The cysts floating on the top of these tubes were collected into a tube mounted with a 150-μm-pore screen, and eggs were released by crushing the cysts on a 150-μm-aperture sieve with a rubber stopper mounted on a motor ( Faghihi and Ferris, 2000). The eggs were cleaned and separated from debris by centrifugation in a 38% (w/v) sucrose solution for 5 min at 1500×g to remove most of the remaining debris. The collected eggs were transferred onto a 38-μm-aperture sieve, rinsed with sterile water, treated with SCQ antibiotic solution (100 ppm streptomycin sulfate, 50 ppm chlorotetracycline, and 20 ppm 8-quinolinol) for 24 h at 4 °C. They were then placed on a 35-μm-aperture nylon cloth on a screen immersed in 4 mM ZnCl2 solution to hatch J2s. The temperature of the containers was maintained at approximately 22–24 °C. The viability and concentration of J2s was determined under an inverted microscope. Suspensions of J2s were then inoculated into approximately 5 cm deep holes dug adjacent to the root systems of each seedling with a 1 mL pipet tip. Approximately 600 juveniles per plant for a total of 3000 juveniles per pot were inoculated 1 week after planting.