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 yello wing virus (BMYV), Brassica yellows virus (BrYV, synonym BWYV) and Beet chl orosis virus (BChV) have been characterised from the genus Polerovirus. Unt il recently, no available tools were available to allow accurate and reliab le distinction of the three species. Based on previous nucleotide sequence alignments and phylogenetic studies, we show that the use of molecular meth ods enabled the discrimination of these three beet Polerovirus species, but with differences in efficiency and specificity. Primers CP + and CP - enco mpassing ORF-3, which encodes the coat protein, allowed the amplification b y RT-PCR of a fragment of 563 bp for all isolates. Molecular methods such a s SSCP or RFLP were able to discriminate these fragments by utilizing the d ifferences in sequence. However, SSCP is a highly sensitive technique and w as not suitable for the distinction of the Polerovirus species, because all isolates tested displayed a unique pattern. Analysis of the ORF3 RT-PCR pr oducts, 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 diff icult. Primers were designed to amplify specifically sequences correspondin g 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 poss ible in infected plants. The multiplex RT-PCR method provides a reliable an d highly sensitive method to detect and identify viral species and will be of great interest for epidemiological studies of beet poleroviruses. (C) 20 00 Elsevier Science B.V. All rights reserved.