A RT–PCR Assay Combined with RFLP Analysis for Detection and Differentiation of Isolates of Pepino Mosaic Virus (PepMV) from Tomato

Abstract

The partial nucleotide sequence of the RNA polymerase gene from one isolate of Pepino mosaic virus (PepMV) was determined. Phylogenetic and distance analysis indicated that this isolate was related to other isolates of PepMV previously reported. To develop a method for detecting PepMV by reverse transcriptase–polymerase chain reaction (RT–PCR), a pair of primers was designated from RNA polymerase sequences. RT–PCR with RNA from a large number of tomato samples with PepMV symptoms, positive controls of PepMV, weed samples containing PepMV, and other potexviruses as negative controls confirmed the specificity of the primers. Restriction endonuclease digestion of the RT–PCR products distinguished three restriction fragment length polymorphism (RFLP) types. The majority of isolates were included within type P1, which correspond with the PepMV isolates found in Europe. This type and type P2, which corresponded with the original PepMV isolated from Solanum muricatum, appear more closely related to each other than to type P3. Type P3 had completely different RFLPs from the other two types studied. It may represent a further line within the PepMV virus. The RT–PCR–RFLP assay is proposed as a rapid and easy method to detect and identify new isolates of the PepMV virus.