Three seeds of each genotype are planted in Ray Leach Cone-tainers (20.6 cm long cones) filled with fumigated sandy loam soil to within 5 cm of the top and then covered with 2.5 cm of fumigated sand.
Ten Cone-tainers each of a susceptible and a resistant standard cultivar are included in each test. Forty-nine Cone-tainers are placed in a RL-98 tray, filling every other row of the tray. The trays (49) are placed on a greenhouse bench under supplemental lighting provided by 400-watt metal halide lamps and under an automatic irrigation system. Seven to ten days after planting, the plants are thinned to one seedling per Cone-tainer and inoculated with 3,000 Root-knot nematode eggs collected with 0.5% NaOCl (10% Clorox). The inoculum (3-5 ml depending on egg concentration) is placed with a digital dispensing pump in the soil at a depth of 2-3 cm. The plants are watered manually for 2 days following inoculation before turning on the automatic irrigation system. All plants are fertilized weekly with 20-20-20 (N=20%, P=8.7%, K=16.6%) solution.
Thirty days after inoculation, roots from a few standard checks are examined for galls to assess whether to begin the process of evaluating the test. For evaluation, the shoots are excised and the root systems are removed from the Cone-tainers and washed free of soil. Screening of advanced breeding lines, the total number of galls per root system is counted. All other studies, the number of galls on the remainder of the susceptible and resistant check plants are used to develop a gall index of 1 (<10 galls per plant) to 5 (>90 galls per plant) for evaluating the genotypes.
