A primer-introduced restriction analysis-PCR-based method to analyse Pepper mild mottle virus populations in plants and field soil with respect to virus mutations that break L3 gene-mediated resistance of Capsicum plants

One or a few nucleotide changes in the coat protein gene reportedly confer Pepper mild mottle virus (PMMoV) with the ability to overcome L3 gene-mediated resistance in Capsicum plants. Primer-introduced restriction analysis-PCR (PIRA-PCR) was set up to detect four known resistance-breaking mutations. Each mutation from the L3-breaking PMMoV strains was successfully detected by reverse transcription-PCR amplification of the viral coat protein gene, PCR amplification of the regions containing the mutations with restriction site-introducing primers, followed by restriction analysis. Since PIRA-PCR primers were designed such that only PCR products from L3-breaking PMMoV strains can be digested by appropriate restriction enzymes, L3-breaking strains could be detected when they comprised no more than 20% of the mixture with non-L3-breaking strain. Using this PIRA-PCR-based method, L3-breaking PMMoV was detected in field soil samples taken from the base of both diseased and non-diseased plants harbouring L3. The results show that PIRA-PCR is useful to quickly detect L3-breaking PMMoV strains not only in Capsicum plants harbouring the L3 resistance gene but also in field soil, which serves as a reservoir of infectious viruses.