Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction

The three most economically damaging ilarviruses affecting stone fruit tree s on a worldwide scale are the related Prunus necrotic ringspot virus (PNRS V), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic mol ecular hybridization and multiplex reverse-transcription polymerase chain r eaction (RT-PCR) methodologies were developed that could detect all these v iruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed whi ch was used in conjunction with three virus-specific sense primers. The amp lification efficiencies for the detection of the three viruses in the multi plex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to ampl ify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring natu rally in single or multiple infections. For ApMV isolates, differences in t he electrophoretic mobilities of the PCR products were observed. The nucleo tide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nu cleotide deletion in the sequence of isolates showing the faster electropho retic mobility. To our knowledge, this is the first report on the simultane ous detection of three plant viruses by multiplex RT-PCR in woody hosts. Th is multiplex RT-PCR could be a useful time and cost saving method for index ing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.