RT-PCR using redundant primers to detect the three viruses associated withcarrot motley dwarf disease

A method was developed for detecting and distinguishing the viruses associa ted with carrot motley dwarf (CMD) disease, i.e. Carrot mottle umbravirus ( CMoV), Carrot red leaf virus (CRLV) and the virus known as carrot red leaf virus-associated RNA. (CRLVaRNA). Redundant primers were made that targeted the RNA-dependent RNA polymerase (RdRp) gene in all available sequences of umbraviruses and in a subset of polerovirus genomes, and specific and redu ndant primers were made to target the same gene in CRLVaRNA. By using these primers in RT-PCR reactions, cDNAs were amplified from total RNA isolated from Belgian parsley with CMD disease. The cDNAs were sequenced and all ana lysis of this data showed that the plants contained all three viruses. RT-P CR assays were optimized for the simultaneous detection of the three viruse s in infected parsley and chervil plants and in individual viruliferous aph ids. This is the first report of a natural infection of CRLVaRNA in a plant other than carrot and the first report of this virus outside the USA. The study also confirmed that the urnbravirus in parsley with CMD disease it; C MoV, and that this virus is distinct from carrot mottle mimic umbravirus (C MoMV), which is also associated with CMD, but apparently not in Europe. (C) 2000 Elsevier Science B.V. All rights reserved.