DiscussionTBTD causes serious symptoms on tobacco leaves, thus reduc-ing greatly the quality of the crop (Cole, 1962; Gates, 1962; Liet al., 2005b). The disease agents are transmitted easily by greenpeach aphids in the fields. Once the disease is introduced, it spreadsquickly throughout the fields and causes irreversible crop loss. Oneeffective way to prevent the disease is to use rapid and sensitivemethods to detect the disease agents and establish effective waysto monitor the population of the vector aphids in the fields. Nucleicacid-based techniques, especially multiplex RT-PCR, provide fast,reliable and cost-effective methods to detect the four TBTD agents.This study developed a one-step multiplex RT-PCR assay for simul-taneous detection the four causal agents of TBTD. The assay isspecific, sensitive, rapid, and cost-effective.