Inoculation Method for Studying Early Responses of Glycine max to Heterodera glycines

An inoculation technique was developed for studying molecular responses of soybean to the soybean cyst nematode (Heterodera glycines). Effect of inoculum age (0-7 days after eggs were released from cysts) and inoculation site (meristem, elongation, or differentiation zone) on infection were tested on four soybean genotypes. Two genotypes (PI 437654 and cv. Peking) were resistant and two (cv. Essex and cv. Hutcheson) were susceptible to race 3 of H. glycines. Inoculum consisting of second-stage juveniles (J2) was prepared by gently agitating nematode eggs at 75 revolutions per minute at 28 degrees C for various intervals. Infection rates were monitored cytologically. The most consistent infection rate was obtained with 48-hour-old inoculum containing more than 80% J2. More than 100 juveniles/root were observed after inoculation with the 48-hour-old inoculum placed at the root elongation zone, in both resistant and susceptible soybeans. Horizontal orientation of roots during inoculation, the use of concentrated J2 inoculurn (500 J2 in 125 mul/root), and restriction of inoculum to the root elongation zone facilitated synchronous root infection.