Improved detection and differentiation of poleroviruses infecting beet or rape by multiplex RT-PCR

Three distinct species of virus inducing yellowing of beet, Beet mild yellowing virus (BMYV), Brassica yellows virus (BrYV, synonym BWYV) and Beet chlorosis virus (BChV) have been characterised from the genus Polerovirus. Until recently, no available tools were available to allow accurate and reliable distinction of the three species. Based on previous nucleotide sequence alignments and phylogenetic studies, we show that the use of molecular methods enabled the discrimination of these three beet Polerovirus species, but with differences in efficiency and specificity. Primers CP+ and CP- encompassing ORF-3, which encodes the coat protein, allowed the amplification by RT-PCR of a fragment of 563 bp for all isolates. Molecular methods such as SSCP or RFLP were able to discriminate these fragments by utilizing the differences in sequence. However, SSCP is a highly sensitive technique and was not suitable for the distinction of the Polerovirus species, because all isolates tested displayed a unique pattern. Analysis of the ORF3 RT-PCR products, digested with SmaI, RsaI and AccI restriction enzymes revealed four distinct patterns specific for the three species. However, point mutations can alter the RFLP patterns, making the interpretation of the results difficult. Primers were designed to amplify specifically sequences corresponding to ORF-0 of the three viral species. By using the three new sets of ORF-0 specific primers and CP+/CP- primers in a single multiplex RT-PCR, the detection and discrimination of the three beet Polerovirus species was possible in infected plants. The multiplex RT-PCR method provides a reliable and highly sensitive method to detect and identify viral species and will be of great interest for epidemiological studies of beet poleroviruses.