Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNAi-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, stp1 and stp2 in tomato seedlings triggers corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponds directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observe no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato, and potentially other crop plants.