FIG. 4.

Real-time PCR analysis of PSTVd levels in infected plant species. Total RNA was isolated from leaf tissues by using the CONCERT plant RNA purification reagent, followed by treatment with DNase and purification by using a QIAGEN RNeasy RNA cleaning protocol. RNA samples were subjected to RT and real-time PCR as described in Materials and Methods. Samples a to d correspond to templates from 0.2 μg of total RNA, samples e and f were from 1 μg of total RNA. Curves: a, Lycopersicon esculentum; b, Anthemis arvensis; c, Chamomilla recutita; d, Amaranthus retroflexus; e, Veronica agrestis; f, Erodium cicutarium. Amplified product in reactions a to e corresponded to the specific 101-bp fragment. No amplification product was detected in reaction f. The threshold level of fluorescence, as designated by the arrow, was set to a value of 1,028.