Sample collection and measurement of lesion length for pot and field experiments

Three randomly selected whole plants were harvested (during a 2-h period beginning at 10 am) from each of the three replicate pots or plots—one plant for each of three different stages of maize development. These stages were the silk, blister and dough stage corresponding to 7, 14 and 21 days after the inoculation with F. verticillioides, respectively. Leaves from these plants were used to determine the activities of defense-related enzymes, protein concentration and protein profile (by SDS-PAGE). For the study of the expression of PR and defense-related genes by RT-qPCR, one fully expanded leaf from a randomly selected plant was harvested from each of the three replicate pots or plots 72 h after inoculation.

At the dough stage (21 days after inoculation), three plants from each pot and five plants from each plot were harvested and scored for disease reduction. The disease rating—average lesion length per pot/plot—was determined by splitting the stalks longitudinally and measuring the length (in cm) of the visible necrotic lesion²⁸.