Detection and differentiation of serologically cross-reacting tobamoviruses of economical importance by RT-PCR and RT-PCR-RFLP

A procedure involving reverse transcription followed by the polymerase chain reaction (RT-PCR) using a single primer pair was developed for the detection of five tobamovirus species which are related serologically. Either with a subsequent restriction, enzyme analysis (RT-PCR-RFLP) or with a RT-PCR using species specific primers the five species can be differentiated. To differentiate those species by serological means is time consuming and might give ambiguous results. With the example of the isolate OHIO V, which is known to break the resistance in a selection of Lycopersicon peruvianum, the suitability of the RT-PCR-RFLP technique to detect variability at the species level was shown. In sequence analysis 47 codons of the coat protein gene of this isolate were found to be mutated compared to a tobacco mosaic virus (TMV) coat protein gene sequence. Forty of these mutations were silent and did not change the amino acid sequence. Both procedures are suitable to detect mixed infections. In addition, the RT-PCR-RFLP give information on the relative amounts of the viruses that are present in a doubly infected plant. The RT-PCR-RFLP using general primers as well as the RT-PCR using species specific primers were proven to be useful for the diagnosis and control of the disease and will be helpful for resistance breeding, epidemiological investigations and plant virus collections. (C) 2002 Published by Elsevier Science B.V.