Apple stem pitting virus detection from dormant pome fruits by RTPCR

Abstract

Apple stem pitting foveavirus is a major latent virus of pome fruits, and thus its reliable diagnostics is a crucial step in virus-free material production and prevention of ASPV spread by infected graftwoods. Thus it would be very convenient a relatively simple, accurate and rapid method in routine testing. The possibility of reliable Apple stem pitting virus detection in pome fruit species by RTPCR during dormancy was assessed. The following commercial kits: QuickPrep(TM) Total RNA Extraction Kit and Ready-To-Go(TM) You-Prime First-Strand Beads Kit (Amersham Pharmacia Biotech Inc., USA), were used for the extraction of total RNA from the bark of annual shoots and conducting RT-PCR, respectively. The application of QuickPrep(TM) Total RNA Extraction Kit was adjusted to the extraction of total RNA from the plant material. The amplification of ASPV-specific DNA fragments of the expected size (291 bp) was obtained from woody and herbaceous indicator plants infected with different isolates originated from apple, pear and quince. Specific DNA fragments were also amplified directly from different apple and pear cultivars infected with ASPV. The ASPV was detected using total RNA extracted from 30 mg or 60 mg of bark as a template and primer pair (ASPR5 and ASPF1, designed by Malinowski et al. 1998) with target sequences in the first open reading frame of ASPV genome. A number of ASPV isolates were detected regardless of the plant host species.