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

Abstract

A method was developed for detecting and distinguishing the viruses associated 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 redundant 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 an analysis of this data showed that the plants contained all three viruses. RT-PCR assays were optimized for the simultaneous detection of the three viruses in infected parsley and chervil plants and in individual viruliferous aphids. 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 umbravirus in parsley with CMD disease is CMoV, and that this virus is distinct from carrot mottle mimic umbravirus (CMoMV), which is also associated with CMD, but apparently not in Europe.