Detection and identification of <i>Apple stem pitting virus</i> and <i>Apple stem grooving virus</i> affecting apple and pear trees in Egypt

Abstract

Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) are economically important and infect either individually or in mixed infection commercial apple and pear cultivars causing yield loses. Young green bud and/or base of petiole were collected from naturally infected apple and pear trees from different locations in Egypt. Both viruses were detected frequently in apple and pear samples. A total of 420 trees from 9 different orchards were tested using one-step RT-PCR; 13% and 17% of these samples were infected with ASPV and ASGV, respectively. Mixed infection with both viruses occurred in 4% of the tested trees. ELISA was reliable for detection of ASGV but not ASPV. Total RNA for one-step RT-PCR was isolated from 100 mg fresh affected apple and pear leaf tissue using Qiagen RNeasy plant mini-kit (Qiagen, Crawley, UK), according to the manufacturer’s instructions. The one step-RTPCR method was performed using ASPV and ASGV-specific primers for each virus. A 316 bp fragment for ASPV and 524bp fragment for ASGV were amplified and detected by gel electrophoresis analysis which indicated the presence of ASPV and ASGV in affected apple and pear cultivars. Southern blot hybridization of the amplified products to digoxigenin (DIG)-labeled cDNA probe for ASPV or ASGV confirmed the results obtained by electrophoresis analysis. No product was detected in amplified extracts of uninfected apple and pear samples. The detection of ASPV and ASGV by one step-RT-PCR assay was successful and appears useful for testing pome fruit germplasm in quarantine and budwood in certification programs.

Keywords: Apple and pear, ASPV, ASGV, virus detection, One step RT-PCR, Southern blot hybridization