The amplification efficiency for each set of WRKY real time PCR primers was determined using serial diluted cDNA or genomic DNA (110 nge11 pg) extracted from newly emerged leaf tissue using a GenElute plant genomic DNA miniprep kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. For the primers of the SA and JA/ethylene-based defense pathway indicator genes (ZmNPR1, ZmPR-1, and ZmERF1), five-fold serial dilutions from 29 ng to 46.4 pg were used for amplification efficiency determination.