Nematode extraction and counting

Nematodes were extracted from the soil using the sieve and sucrose centrifugation method. Each soil sample was thoroughly mixed and a 200 cm³ sub-sample was used for extraction, identification, and quantification of the nematodes. The infested soil was placed in a bucket of running water until the soil was covered by at least two times its volume. The solution was then mixed vigorously until the soil was sufficiently dispersed and left untouched to settle for 3 min. The liquid supernatant was then poured through a 90 μm sieve nested onto a 36 μm sieve. The 36 μm sieve was washed thoroughly with water until as much clay and other fine particles were washed out of the sieve. The remaining sample with the nematodes was then washed into a 50 ml centrifuge tube. Centrifugation was carried out at 1700 rpm for 5 min in an M 23i benchtop centrifuge (Jouan-Thermo Scientific, U.S.A.). The supernatant was discarded and if necessary, successive samples centrifuged until a final pellet obtained from the collective population of nematodes. The tubes were filled with sucrose solution and stirred with a spatula to break up the pellet, afterwards the sample was spun at 1,000 rpm for 1 min. The supernatant was poured onto a 36 μm sieve and thoroughly washed with tap water to get rid of the sucrose, bringing the final volume with the nematodes to 10 ml in labeled vials. Nematodes were extracted from infested tomato roots, using modified Baermann funnel method. The roots were chopped with a pair of scissors and about 10 g was blended for two five second bursts. The partially macerated roots were transferred into a glass funnel lined with two ply tissue paper placed on a wire mesh. Tap water was poured gently into the funnel in which the mesh was placed until the tissue became moist. The set-up was left for 48 h for nematodes to migrate to the bottom of the tubings and collected into beakers. Each nematode suspension was separately topped with tap water to 300 ml for standardization.