APPLE DIMPLE FRUIT VIROID: SEQUENCE VARIABILITY AND ITS SPECIFIC DETECTION BY MULTIPLEX FLUORESCENT RTPCR IN THE PRESENCE OF APPLE SCAR SKIN VIROID
F. Di Serio, M. Malfitano, D. Alioto, A. Ragozzino, R. Flores
Apple dimple fruit viroid (ADFVd) causes a severe fruit disorder in several apple cultivars. The viroid was discovered a few years ago, and only a limited number of sequence variants has been reported so far. In this work, the sequence variability of two ADFVd field isolates from two commercial apple cultivars was studied. Sequencing of 18 full-length cDNA clones revealed five new sequence variants, all adopting a similar quasi-rodlike secondary structure of lowest free energy. Sequence comparison showed nine polymorphic positions distributed in different regions of the ADFVd molecule. Since symptoms of apple dimple fruit disease resemble those of dapple apple induced by Apple scar skin viroid (ASSVd), and it has been experimentally demonstrated that these two viroids may co-exist in the same plant, a fast and sensitive method for their rapid detection and discrimination is needed. We report here a method that meets such needs. Total nucleic acid minipreparations from symptomatic fruits, obtained by extraction with buffer-saturated phenol and further treatment with a silica-gel capture system, were used as templates for the simultaneous detection of ADFVd and ASSVd by multiplex fluorescent RT-PCR amplification. The incorporation in the PCR reaction of two viroid-specific primers, each labeled with a different fluorescent dye, simplifies and facilitates distinction of the amplified products, and avoids the use of the mutagenic and cancer inducing agent ethidium bromide for gel staining.